The plant pathogen Pantoea ananatis produces N-acylhomoserine lactone and causes center rot disease of onion by quorum sensing.
ABSTRACT A number of gram-negative bacteria have a quorum-sensing system and produce N-acyl-l-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule. Pantoea ananatis is reported as a common colonist of wheat heads at ripening and causes center rot of onion. In this study, we demonstrated that P. ananatis SK-1 produced two AHLs, N-hexanoyl-l-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). We cloned the AHL-synthase gene (eanI) and AHL-receptor gene (eanR) and revealed that the deduced amino acid sequence of EanI/EanR showed high identity to those of EsaI/EsaR from P. stewartii. EanR repressed the ean box sequence and the addition of AHLs resulted in derepression of ean box. Inactivation of the chromosomal eanI gene in SK-1 caused disruption of exopolysaccharide (EPS) biosynthesis, biofilm formation, and infection of onion leaves, which were recovered by adding exogenous 3-oxo-C6-HSL. These results demonstrated that the quorum-sensing system involved the biosynthesis of EPS, biofilm formation, and infection of onion leaves in P. ananatis SK-1.
- SourceAvailable from: msk.or.kr[show abstract] [hide abstract]
ABSTRACT: To gain maximal benefit in a competitive environment, single-celled bacteria have adopted a community genetic regulatory mechanism, known as quorum sensing (QS). Many bacteria use QS signaling systems to synchronize target gene expression and coordinate biological activities among a local population. N-acylhomoserine lactones (AHLs) are one family of the well-characterized QS signals in Gram-negative bacteria, which regulate a range of important biological functions, including virulence and biofilm formation. Several groups of AHL-degradation enzymes have recently been identified in a range of living organisms, including bacteria and eukaryotes. Expression of these enzymes in AHL-dependent pathogens and transgenic plants efficiently quenches the microbial QS signaling and blocks pathogenic infections. Discovery of these novel quorum quenching enzymes has not only provided a promising means to control bacterial infections, but also presents new challenges to investigate their roles in host organisms and their potential impacts on ecosystems.The Journal of Microbiology 03/2005; 43 Spec No:101-9. · 1.28 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Erwinia amylovora causes fire blight disease of apple, pear, and other members of the Rosaceae. Here we present the first evidence for autoinduction in E. amylovora and a role for an N-acyl-homoserine lactone (AHL)-type signal. Two major plant virulence traits, production of extracellular polysaccharides (amylovoran and levan) and tolerance to free oxygen radicals, were controlled in a bacterial-cell-density-dependent manner. Two standard autoinducer biosensors, Agrobacterium tumefaciens NTL4 and Vibrio harveyi BB886, detected AHL in stationary-phase cultures of E. amylovora. A putative AHL synthase gene, eamI, was partially sequenced, which revealed homology with autoinducer genes from other bacterial pathogens (e.g., carI, esaI, expI, hsII, yenI, and luxI). E. amylovora was also found to carry eamR, a convergently transcribed gene with homology to luxR AHL activator genes in pathogens such as Erwinia carotovora. Heterologous expression of the Bacillus sp. strain A24 acyl-homoserine lactonase gene aiiA in E. amylovora abolished induction of AHL biosensors, impaired extracellular polysaccharide production and tolerance to hydrogen peroxide, and reduced virulence on apple leaves.Journal of Bacteriology 06/2005; 187(9):3206-13. · 3.19 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: N-acylhomoserine lactones, known as autoinducers (AIs), are widely conserved signal molecules present in quorum-sensing systems of many gram-negative bacteria. AIs are involved in the regulation of diverse biological functions, including expression of pathogenic genes in the plant pathogens Pseudomonas solanacearum, several Erwinia species, and the human pathogen Pseudomonas aeruginosa. A bacterial isolate, Bacillus sp. 240B1, is capable of enzymatic inactivation of AIs. The gene (aiiA) for AI inactivation from Bacillus sp. 240B1 has been cloned and shown to encode a protein of 250 amino acids. Sequence alignment indicates that AiiA contains a "HXHXDH" zinc-binding motif that is conserved in several groups of metallohydrolases. Site-directed mutagenesis showed that conserved aspartate and most histidine residues are required for AiiA activity. Expression of aiiA in transformed Erwinia carotovora strain SCG1 significantly reduces the release of AI, decreases extracellular pectolytic enzyme activities, and attenuates pathogenicity on potato, eggplant, Chinese cabbage, carrot, celery, cauliflower, and tobacco. Our results indicate that the AI-inactivation approach represents a promising strategy for prevention of diseases in which virulence is regulated by AIs.Proceedings of the National Academy of Sciences 04/2000; 97(7):3526-31. · 9.74 Impact Factor
JOURNAL OF BACTERIOLOGY, Nov. 2007, p. 8333–8338
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 189, No. 22
The Plant Pathogen Pantoea ananatis Produces N-Acylhomoserine
Lactone and Causes Center Rot Disease of Onion by
Tomohiro Morohoshi,1* Yuta Nakamura,1Go Yamazaki,2Akio Ishida,2
Norihiro Kato,1and Tsukasa Ikeda1
Department of Applied Chemistry, Utsunomiya University, Utsunomiya, Tochigi 321-8585, Japan,1and Department of
Environmental Science, Kumamoto University, Kumamoto 860-8555, Japan2
Received 4 July 2007/Accepted 29 August 2007
A number of gram-negative bacteria have a quorum-sensing system and produce N-acyl-L-homoserine
lactone (AHL) that they use them as a quorum-sensing signal molecule. Pantoea ananatis is reported as a
common colonist of wheat heads at ripening and causes center rot of onion. In this study, we demonstrated that
P. ananatis SK-1 produced two AHLs, N-hexanoyl-L-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl)-L-
homoserine lactone (3-oxo-C6-HSL). We cloned the AHL-synthase gene (eanI) and AHL-receptor gene (eanR)
and revealed that the deduced amino acid sequence of EanI/EanR showed high identity to those of EsaI/EsaR
from P. stewartii. EanR repressed the ean box sequence and the addition of AHLs resulted in derepression of
ean box. Inactivation of the chromosomal eanI gene in SK-1 caused disruption of exopolysaccharide (EPS)
biosynthesis, biofilm formation, and infection of onion leaves, which were recovered by adding exogenous
3-oxo-C6-HSL. These results demonstrated that the quorum-sensing system involved the biosynthesis of EPS,
biofilm formation, and infection of onion leaves in P. ananatis SK-1.
Quorum sensing is one of the cell-cell communication mech-
anisms depending on cell population density in bacteria (4, 11).
In many gram-negative bacteria, several kinds of N-acyl-L-
homoserine lactone (AHL) have been identified as signal com-
pounds involved in this mechanism and called autoinducers (4,
11). AHLs are synthesized in bacteria by a member of the LuxI
protein family and diffused outside and inside of bacteria.
When AHL concentration increases and reaches a threshold
due to accumulation of AHL derived from each bacterial cell,
AHL receptor protein belonging to the LuxR protein family
binds AHL and regulates expression of many genes responsible
for bioluminescence, production of pigment or antibiotics, and
so on (11). In particular, many gram-negative pathogens con-
trol the expression of virulence factors, which include the se-
cretion of extracellular protease, pectinase, and biosurfactant
and forming biofilm (4).
Many plant pathogens produce AHLs and regulate their
virulence by AHL-mediated quorum sensing (2, 27). Erwinia
carotovora, which causes soft rot diseases on many plant spe-
cies, induces the production of various exoenzymes and plant
tissue maceration by AHLs (3). Pantoea stewartii regulates
exopolysaccharide (EPS) biosynthesis and pathogenicity in
sweet corn by AHLs (28). Erwinia amylovora produces one
AHL and regulates EPS biosynthesis, tolerance to hydrogen
peroxide, and the development of symptoms on apple leaves
(18). Agrobacterium vitis causes necrosis on grape plants and a
hypersensitive-like response on tobacco plants by a quorum-
sensing system (30). In general, AHL-negative mutants show
defects in pathogenicity, so it is expected that disrupting or
manipulating quorum-sensing signals inhibits the expression of
virulence and infection of host cells. Recently, some AHL-
degrading bacteria and enzymes have been reported (7). An
AHL lactonase-encoded gene (aiiA) was cloned from Bacillus
sp. strain 240B1 (6). The expression of aiiA in transformed E.
carotovora significantly attenuates pathogenicity in some crops
(6). Expression of aiiA in E. amylovora impairs EPS production
and reduces virulence on apple leaves (18). Transgenic plants
expressing AHL lactonase exhibited significantly enhanced re-
sistance to E. carotovora infection (5).
P. ananatis is reported as a common colonist of wheat heads
at ripening and causes “center rot” disease in onion plants (1,
9, 29). Center rot was first reported on an onion in Georgia in
1997 and has continued to reduce yields and cause postharvest
losses (9). Onion leaves infected by P. ananatis are usually
collapsed and hang down beside the onion neck (1). For the
treatment of center rot, fixed copper materials tank mixed with
EBDC fungicides are recommended to suppress infection and
spread (1). However, the major virulence factors of P. ananatis
are unknown. Recently, it was reported that P. ananatis strains
produced AHLs. Yoshida et al. revealed that P. ananatis in-
habiting wheat heads produced at least two AHLs, N-hexanoyl-
L-homoserine lactone (C6-HSL) and N-(3-oxohexanoyl)-L-
homoserine lactone (3-oxo-C6-HSL) (29). Pomini et al.
reported that P. ananatis (Serrano 1928) produced three
AHLs, and the major identified substance is C6-HSL (22).
However, these authors did not identify the LuxRI homologs
from P. ananatis strains and elucidate the phenotypes con-
trolled by AHL-mediated quorum sensing. We report here the
identification of the LuxRI homologs, EanRI, and C6-HSL
and 3-oxo-C6-HSL in P. ananatis strain SK-1. We also present
evidence for the involvement of the quorum-sensing system in
* Corresponding author. Mailing address: Department of Applied
Chemistry, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, 321-8585,
Japan. Phone and fax: 81-28-689-6176. E-mail: email@example.com
?Published ahead of print on 7 September 2007.
the regulation of EPS biosynthesis, biofilm formation, and the
infection of onion leaves.
MATERIALS AND METHODS
Bacterial strains, plasmids, compounds and growth conditions. Selected bac-
terial stains and plasmids used in the present study were listed in Table 1.
Escherichia coli and Chromobacterium violaceum were grown at 30°C in Luria-
Bertani (LB) medium (24). P. ananatis was grown at 30°C in tryptic soy broth
(TSB; Becton Dickinson). Solid bacterial media were made by the addition of
agar at a final concentration of 1.5%. Antibiotics were added as required at final
concentrations of 100 ?g of ampicillin/ml, 10 ?g of chloramphenicol/ml, 10 ?g of
colistin/ml, and 10 ?g of gentamicin/ml. AHL standards were synthesized by a
previously described method (14). pKRP14, which carried gene cassettes impart-
ing resistance to the gentamicin (Gmr), was constructed in the present study.
Briefly, a Gmrcassette was amplified from pJN105 by using the following prim-
ers, which contained HindIII restriction sites (underlined) at their 5? ends:
5?-AAGCTTTCGCCTTGCGTATAATATTTGCCC-3? and 5?-AAGCTTTGA
CAATTTACCGAACAACTCCGC-3?. PCR fragments were cut out by HindIII
digestion and inserted into the HindIII-digested pKRP11 for construction of
Identification and characterization of AHLs. AHLs produced by P. ananatis
SK-1 were isolated and purified by a previously described method (17). The AHL
sample was subjected to analytical and preparative thin-layer chromatography
(TLC). TLC analysis was carried out on a C18reversed-phase TLC plate
and standards were spotted onto a TLC plate and developed with 60% (vol/vol)
methanol in water. The air-dried plate was overlaid with LB soft gel (1% agar)
with C. violaceum CV026 biosensor (17) and incubated at 30°C. AHL production
was also assayed by cross-streaking against CV026 biosensor as the AHL bio-
sensor. Briefly, CV026 was streaked at the center of the LB agar plate. The target
bacteria were streaked on the same plate next to CV026 line. Diffusible AHL
produced by the target bacteria induces strain CV026 to produce a purple
Cloning and disruption of chromosomal eanI-eanR locus. Chromosomal DNA
of SK-1 was extracted to construct genomic library by the standard protocol (24).
DNA was digested partially with Sau3AI, and the fragments were inserted into
the BamHI site of cloning vector, pSTV28. The genomic library of SK-1 was
transformed into E. coli DH5?, and we checked the AHL-producing ability by
cross-streaking with a CV026 biosensor. One of the AHL-producing plasmids,
pAN01, was sequenced by using BigDye terminator version 3.1 and an ABI Prism
3100 genetic analyzer (Applied Biosystems). The eanI/eanR locus on the chro-
mosomal DNA of SK-1 was amplified by PCR using the primers 5?-GTAAAA
TCAGTACAGGATAGCCGTGAGGGC-3? and 5?-TAAAGGAGGACAATC
AGGTGTGGGAAAGCG-3? and cloned into pGEM-T Easy cloning vector for
construction of pAN02. To disrupt the eanI gene, pAN02 was digested with
HindIII and inserted the 900-bp Gmrcassette from HindIII-digested pKRP14.
The eanI::Gmrregion was cut out by EcoRI digestion and inserted into the MunI
site of pGP704Sac38 for construction of pGP704EIG. To disrupt the eanR gene,
pAN02 was digested with BglII, and the Gmrcassette was inserted from BamHI-
digested pKRP14. The eanR::Gmrregion was cut out by EcoRI digestion and
inserted into the MunI site of pGP704Sac38 for construction of pGP704ERG.
Disruption of chromosomal eanI and eanR in strain SK-1 was performed by
bacterial conjugation and homologous recombination (19). Conjugation was
conducted between SK-1 and E. coli S17-1 ?pir carrying pGP704EIG or
pGP704ERG. The chromosomal disruption of eanI and eanR was checked by
PCR using the same primers, and the insertion mutants of eanI and eanR were
designated SK-02I and SK-05R, respectively.
Promoter assay. The putative promoter region of eanR was amplified by PCR
using the primers 5?-TAAAGGAGGACAATCAGGTGTGGGAAAGCG-3?
and 5?-GTTTAAAGGCGGTAAGGATAACCGGATCGG-3? and cloned into
pGEM-T Easy. The putative promoter region was cut out by SphI and SalI
digestion and cloned into the SphI and SalI sites of vector pQF50 for the
construction of pQF50ER. To construct the EanR expression plasmid, the pro-
moter-less eanR gene was amplified by PCR using the primers 5?-ATCGTTAA
GTAAAAGAAGCAGCATGGAGCC-3? and 5?-TACTCAAACGGTCCGGA
TGGCAAATCAGCG-3? and cloned into pGEM-T Easy. The promoter-less
eanR was cut out by EcoRI digestion and ligated with EcoRI-digested pJN105 for
TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmidDescriptiona
Natural isolate from Shirakawa river in Kumamoto prefecture (Japan)
SK-1 derivative, eanI::Gmr
SK-1 derivative, eanR::Gmr
F?supE44 ?lacU169 (?80 lacZ?M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1
thi pro hsdR hsdM?recA RP4 2Tc::Mu-Km::Tn7
ATCC 31532 derivative, cviI::Tn5xylE; KmrSmr
Cloning vector; Cmr
Cloning vector; Apr
6.0-kb Sau3AI fragment from SK-1 genomic DNA in pSTV28
pGEM-T easy containing eanI-eanR locus
pBR322 derivative with R6K ori, mob RP4, polylinker from M13 tg131
containing sacB; Apr
pGP704Sac38 containing eanI::Gmr
pGP704Sac38 containing eanR::Gmr
pBBR1MCS-5 with araC and PBADregions before the polylinker; Gmr
pJN105 containing eanR
lacZ transcriptional fusion vector; Apr
pQF50 containing eanR promoter.
Cloning vector; KmrApr
pKRP11 derivative containing the Gmrcassette
aCmr, chloramphenicol resistance; Smr, streptomycin resistance; Apr, ampicillin resistance; Kmr, kanamycin resistance.
8334 MOROHOSHI ET AL.J. BACTERIOL.
the construction of pJN105ER. Both pQF50ER and pJN105ER were trans-
formed into E. coli DH5? for the promoter assay. The DH5? reporter strain was
grown for 15 h and inoculated into 4 ml of fresh LB medium (1% inoculum).
AHLs were added into the subculture at a final concentration of 10 ?M. Arabi-
nose was used for the induction of expression of EanR at a concentration of
0.4%. After incubation for 20 h, ?-galactosidase activity was measured by using
a Galacto-Light Plus kit (Tropix) as described previously (16). The results are
given in units of ?-galactosidase activity relative to the optical density at 600 nm.
Detection of EPS biosynthesis. EPS biosynthesis was evaluated by a previously
described method (28). P. ananatis strains were streaked onto the TSB agar
plate. After incubation for 24 h at 30°C, colonies producing EPS have a mucoid
appearance, whereas those deficient in EPS have a nonmucoid phenotype. Ex-
periments were repeated at least three times.
Biofilm formation assay. Biofilm formation was determined by the previously
described method with slight modification (15). The full-grown cultures of P.
ananatis strains were diluted 100-fold in the TSB medium, and 200 ?l of the
dilution was added to each well of a 96-well polypropylene microtiter plate
(Corning, Inc.). After incubation at 30°C for 20 h, 25 ?l of a 1% crystal violet
solution was added to each well. The plates were incubated at room temperature
for 15 min and rinsed with distilled water. The crystal violet was dissolved in 200
?l of 95% ethanol, and biofilm formation was analyzed at 570 nm by using a
Spectra Max 250 spectrophotometer (Molecular Devices).
Plant infection assays. Pathogenicity of P. ananatis strains was determined on
onion leaves as described previously with slight modifications (10). Briefly, a
sterile needle was dipped into the bacterial colonies on TSB agar plates grown
for 24 h. The needle was then inserted under the epidermis of a leaf. P. ananatis
strains were inoculated at two sites per leaf. Inoculated leaves were incubated at
room temperature and observed for the development of symptoms. All infection
assays contained at least two leaves per treatment, and experiments were per-
formed at least twice.
Nucleotide sequence accession numbers. The nucleotide sequences of the 16S
rRNA gene and the eanI/eanR locus from the strain SK-1 have been deposited in
the DDBJ/EMBL/GenBank databases under accession numbers AB304809 and
RESULTS AND DISCUSSION
P. ananatis SK-1 produces AHLs. P. ananatis SK-1 was iso-
lated from the Shirakawa River in Kumamoto Prefecture (Ja-
pan). The nucleotide sequence of the 16S rRNA fragment
from SK-1 showed 99.9% identity with that of P. ananatis strain
LMG 20103 (accession no. AF364847). Morphological and
biochemical test results were evaluated according to Bergey’s
Manual of Determinative Bacteriology (12). These test results
also suggested that strain SK-1 belonged to P. ananatis (data
not shown). We screened for the AHL production of SK-1 by
cross-streaking against C. violaceum CV026 as the AHL bio-
sensor. Since SK-1 stimulated violacein production of the
CV026 biosensor, it revealed that SK-1 had the ability to pro-
duce AHLs (Fig. 1A). We also checked the structure of pro-
duced AHLs by the TLC analysis. AHLs were extracted with
dichloromethane and fractionated by TLC. TLC-overlaid
CV026 biosensor revealed two AHL spots in the culture su-
pernatants of SK-1. After comparison with AHL standards,
these spots corresponded with C6-HSL and 3-oxo-C6-HSL
(Fig. 1B). In a previous report, P. ananatis inhabiting wheat
heads produces C6-HSL and 3-oxo-C6-HSL (29). It was note-
worthy that a wide range of P. ananatis strains generally pro-
duced C6-HSL and 3-oxo-C6-HSL.
Cloning and characterization of the luxI and luxR homologs
eanI and eanR. For cloning the AHL synthase gene, we con-
structed a genomic library of SK-1 based on pSTV28 cloning
vector. Approximately 2,000 transformants were screened for
the presence of a luxI homolog by toothpicking colonies and
cross-streaking them against the CV026 biosensor. One clone
was able to induce the production of violacein in CV026. Plas-
mid DNA extracted from the positive clone contained 6.0-kb
DNA fragment and was designated pAN01. The nucleotide
sequence of the pAN01 revealed the presence of luxI and luxR
homologs. These luxI and luxR homologs were designated eanI
(for E. ananatis luxI) and eanR (for E. ananatis luxR), respec-
tively. The putative gene product of eanI (EanI) encoded a
210-amino-acid protein and showed 91.9% identity with EsaI
from P. stewartii and 44.3% identity with CarI from E. caroto-
vora (Fig. 2A). The previous report indicated that P. stewartii
produced 3-oxo-C6-HSL as a sole AHL compound (28). Al-
though EsaI from P. stewartii showed high homology with
EanI, EsaI did not produce C6-HSL. EanI and EsaI shared
well-known conserved amino acid residues that have been
demonstrated to be important to AHL synthase (data not
shown) (21). A comparative study of EanI and EsaI could not
explain the reason for the structural difference of AHLs pro-
duced EanI and EsaI. The putative gene product of eanR
(EanR) encoded 249 amino acids and showed 95.2% identity
with EsaR from P. stewartii and 32.8% identity with CarR from
E. carotovora (Fig. 2B). The eanR promoter region contained
putative ?10 and ?35 sequences, and a 20-bp imperfect in-
verted repeat spanned the ?10 region (Fig. 3A). This inverted
repeat sequence can be observed at the promoter region of
luxR homolog in P. stewartii (28) and Serratia marcescens (13).
These inverted repeats were very similar to the lux box con-
sensus sequence (Fig. 3B). Thus, the inverted repeat sequence
in the eanR promoter region is thought to represent the bind-
ing site of LuxR homolog.
EanR acts as a negative regulator. To analyze the function
of EanR for the activation of the lux box-like sequence (ean
box), the upstream region of eanR was amplified by PCR and
cloned upstream of the promoterless lacZ gene of reporter
plasmid pQF50. We monitored the promoter activity of the ean
box in E. coli DH5? carrying the EanR expression plasmid
pJN105ER and the ean box-lacZ plasmid pQF50ER. The ex-
pression of eanR on the pJN105ER was controlled by the PBAD
promoter. The ean box promoter was strongly activated with-
out arabinose but only at a very low level with 0.4% arabinose
FIG. 1. Identification and characterization of AHLs produced by P.
ananatis SK-1. (A) Cross-streaks of SK-1 and its mutants against C.
violaceum CV026. Diffusible AHL production by SK-1 and SK-05R
induced CV026 biosensor to produce a violacein. (B) TLC analysis of
AHLs produced by strain SK-1. AHLs were visualized as pigments
produced by CV026 biosensor and identified after comparison with the
AHL standards C6-HSL and 3-oxo-C6-HSL.
VOL. 189, 2007 QUORUM SENSING IN P. ANANATIS 8335
(Fig. 4). This result suggested that the activation of the ean box
sequence was negatively regulated by EanR. To confirm
whether the addition of AHLs results in derepression of ean
box, various kinds of AHLs were added at a concentration of
10 ?M. The ean box promoter was activated by C6-HSL and
C8-HSL, and their 3-oxo-substitution (Fig. 4). 3-Oxo-substi-
tuted AHLs demonstrated higher activity than 3-oxo-unsubsti-
tuted AHLs (Fig. 4). In the case of P. stewartii, EsaR also
worked as a negative regulator of AHL-mediated quorum
sensing (28). EsaR binds the lux box-like sequence in the ab-
sence of AHL and blocks the transcriptional activity of RNA
polymerase (26). In addition, the ability of EsaR to bind to its
FIG. 2. Multiple alignments of EanI (A) and EanR (B). Gray and black shading indicates similar and identical amino acids, respectively.
Sequence alignment was performed with CLUSTAL W (http://www.ddbj.nig.ac.jp/search/clustalw-j.html) and presented with Boxshade 3.21
(http://www.ch.embnet.org/software/BOX_form.html). The sequences used in the alignments with EanI were EsaI from P. stewartii (accession no.
L32183) and CarI from E. carotovora (X74299). The sequences used in the alignments with EanR were EsaR from P. stewartii (L32184) and CarR
from E. carotovora (AF041840).
FIG. 3. (A) Nucleotide sequence analysis of the upstream region of eanR. Putative ?35 and ?10 promoter sequences, the ribosome binding
site (RB), and the translation initiation site are shown in boldface type. The inverted repeat exhibiting similarity to lux box was underlined.
(B) Nucleotide sequence comparison of consensus lux box sequences (13) and the upstream region of luxR homolog in P. ananatis SK-1, P. stewartii
DC283, and S. marcescens SS-1. Conserved sequences are shown in boldface type.
8336MOROHOSHI ET AL.J. BACTERIOL.
DNA recognition site is antagonized by the presence of 3-oxo-
C6-HSL (26). Our data suggested that EanR might behave in
a fashion similar to its closest homolog, EsaR, and was likely to
bind the ean box promoter in the absence of AHLs.
AHL synthesis requires eanI but not eanR. To determine
whether eanI and eanR are required for AHL synthesis, we
disrupted the genomic eanI and eanR genes in SK-1. The Gmr
gene was inserted to the HindIII or BglII site of the eanI/eanR
locus for construction of eanI and eanR mutants, respectively.
When cross-streaked against CV026 biosensor, the eanR mu-
tant SK-05R showed obvious AHL-producing activity, as well
as the SK-1 parent strain, but the eanI mutant SK-02I did not
produce any AHLs (Fig. 1A). This result demonstrated that
the eanI gene was necessary for the production of both C6-
HSL and 3-oxo-C6-HSL, and the expression of eanI was not
regulated by EanR. In P. stewartii, esaI is expressed constitu-
tively and not regulated by EsaR (28). The lux box-like se-
quence was absent in the upstream region of eanI, as well as
esaI (data not shown). It was assumed that eanI was also
EPS biosynthesis and biofilm formation require eanI or
AHLs. In a previous report on P. stewartii, EPS biosynthesis
was regulated by the esaI gene or 3-oxo-C6-HSL (28). Thus, we
investigated the ability to produce EPS in SK-1 and its mu-
tants. We tested the abilities of these strains to stimulate EPS
biosynthesis on TSB agar plates. After incubation for 24 h,
SK-1 and SK-05R displayed a mucoid phenotype resulting
from the production of EPS, but SK-02I did not (Fig. 5A).
When the TSB agar plate was supplemented with 10 ?M
3-oxo-C6-HSL, the colonies of SK-02I became mucoid, as well
as those of other strains (Fig. 5B). These results demonstrated
that AHLs produced by eanI gene induced the production of
EPS in SK-1. Pathogenesis in P. stewartii correlates with the
ability to produce EPS, and the production of the EPS requires
3-oxo-C6-HSL (28). Thus, it is possible that P. ananatis pro-
duces EPS as a major virulence factor under quorum-sensing
We also tested biofilm formation on a polypropylene plastic
surface. Although SK-1 and SK-05R formed a certain amount
of biomass that adhered to the polypropylene, biofilm forma-
tion of SK-02I was reduced to ca. 60% of the parental level
(Fig. 6). When 3-oxo-C6-HSL was added into each well, the
biofilm formation of SK-02I was increased in a dose-dependent
manner (Fig. 6). This behavior implied that biofilm formation
FIG. 4. Induction of the ean box-lacZ transcriptional fusion in E.
coli DH5? by different AHL compounds. E. coli DH5? carrying
pJN105ER and pQF50ER was grown in the LB medium with various
synthetic AHLs. After 15 h of incubation, the ?-galactosidase activity
was measured. Arabinose was used for the induction of expression of
EanR at a concentration of 0.4%. Ara?indicates activity without
arabinose and AHL. AHL?indicates the result with 0.4% arabinose
and without AHL. The results were reproduced in three repeated
experiments, and error bars indicate standard deviations.
FIG. 5. Production of EPS by wild-type and mutant strains of P.
ananatis SK-1. Strains were streaked onto the TSB agar plates without
(A) or with (B) 10 ?M 3-oxo-C6-HSL. Mucoid and slimy colony
morphology indicated EPS production after incubation at 30°C for
FIG. 6. Quantification of bacteria in biofilm formed on polypro-
pylene plastic by wild-type and mutant strains of P. ananatis SK-1.
Biofilms were allowed to form in a 96-well polypropylene microtiter
dish, stained with crystal violet, and estimated by analysis at 570-nm
absorbance. Six wells of each sample were used for measuring biofilm
formation, and error bars indicate standard deviations.
FIG. 7. Virulence assay of wild-type and mutant strains of P.
ananatis SK-1. Bacterial strains were injected into onion leaves at two
sites per leaf. 3-oxo-C6-HSL was spotted onto the leaves at 100 nmol
per spot. Inoculated onion leaves were incubated at room temperature
for 3 days and monitored for the development of the necrotic
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