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					Microbiology (2004), 150, 2727–2737                                                                          DOI 10.1099/mic.0.27199-0

                                      AlgR functions in algC expression and virulence in
                                      Pseudomonas syringae pv. syringae
                                      Alejandro Penaloza-Vazquez,14 Mohamed K. Fakhr,234 Ana M. Bailey3
                                                   ˜       ´
                                      and Carol L. Bender1
  Correspondence                           Department of Entomology and Plant Pathology1 and Department of Microbiology and
  Carol L. Bender                          Molecular Genetics2, Oklahoma State University, Stillwater, OK 74078, USA                  3
                                                                   ´     ´
                                           Departamento de Ingenierıa Genetica de Plantas CINVESTAV-IPN Unidad Irapuato, Irapuato,
                                           Guanajuato, 36500 Mexico

                                      Pseudomonas syringae pv. syringae strain FF5 is a phytopathogen associated with a rapid
                                      dieback on ornamental pear trees. P. syringae and the human pathogen Pseudomonas aeruginosa
                                      produce the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. In
                                      P. aeruginosa, the response regulator AlgR (AlgR1) is required for transcription of algC and
                                      algD, which encode key enzymes in the alginate biosynthetic pathway. In P. syringae FF5, however,
                                      algR is not required for the activation of algD. Interestingly, algR mutants of P. syringae remain
                                      nonmucoid, indicating an undefined role for this response regulator in alginate biosynthesis.
                                      In the current study, the algC promoter region was cloned from P. syringae pv. syringae strain
                                      FF5, and sequence analysis of the algC promoter indicated the presence of potential binding
                                      sites for AlgR and s54, the alternative sigma factor encoded by rpoN. The algC promoter from
                                      P. syringae FF5 (PsalgC) was cloned upstream of a promoterless glucuronidase gene (uidA), and
                                      the PsalgC–uidA transcriptional fusion was used to monitor algC expression in strains FF5.32
                                      (algR mutant of P. syringae FF5) and PG4180.K2 (rpoN mutant of P. syringae pv. glycinea
                                      PG4180). Expression of the PsalgC–uidA fusion was fourfold lower in both the algR and rpoN
                                      mutants as compared to respective wild-type strains, indicating that both AlgR and s54 are
                                      required for full activation of algC transcription in P. syringae pv. syringae. AlgR from P. syringae
                                      was successfully overproduced in Escherichia coli as a C-terminal translational fusion to the
                                      maltose-binding protein (MBP). Gel shift experiments indicated that MBP–AlgR binds
                                      strongly to the algC promoter region. Biological assays demonstrated that the algR mutant was
                                      significantly impaired in both pathogenicity and epiphytic fitness as compared to the wild-type
                                      strain. These results, along with the gene expression studies, indicate that AlgR has a positive
                                      role in the activation of algC in P. syringae and contributes to both virulence and epiphytic
  Received 30 March 2004              fitness. Furthermore, the symptoms observed with wild-type P. syringae FF5 suggest that this strain
  Revised   16 May 2004               can move systemically in leaf tissue, and that a functional copy of algR is required for systemic
  Accepted 18 May 2004                movement.

INTRODUCTION                                                             a co-polymer of O-acetylated b-1,4-linked D-mannuronic
                                                                         acid and L-guluronic acid, has been reported to function in
Strains of the phytopathogen Pseudomonas syringae, which
                                                                         the virulence of P. syringae by facilitating dissemination of
are subdivided into 50 pathovars according to host range,
                                                                         the bacterium in planta and by enhancing epiphytic fitness
induce a wide variety of symptoms on plants including leaf
                                                                         (Keith et al., 2003; Yu et al., 1999). Alginate biosynthesis has
spots, blights and cankers (Hirano & Upper, 2000). Alginate,
                                                                         been extensively studied in Pseudomonas aeruginosa, where
                                                                         it functions as a major virulence factor in strains infecting
3Present address: Dept of Veterinary and Microbiological Sciences,       the lungs of cystic fibrosis (CF) patients (Lyczak et al., 2002).
North Dakota State University, Fargo, ND 58105, USA.                     The alginate genes are normally silent in P. aeruginosa but
4These two authors contributed equally to this work.                     are specifically activated during growth of this organism in
Abbreviations: ABS, AlgR-binding sites; CF, cystic fibrosis; GUS,         the lungs of CF patients. Activation involves two critical
glucuronidase; MBP, maltose-binding protein.                             promoters, PalgC (Zielinski et al., 1991, 1992) and PalgD
The GenBank accession number for the sequence reported in this           (Deretic et al., 1987). The algC and algD promoters, while
paper is AY575079.                                                       mapping at two separate locations on the P. aeruginosa

0002-7199 G 2004 SGM          Printed in Great Britain                                                                               2727
A. Penaloza-Vazquez and others

chromosome, are nevertheless both responsive to environ-            show a role for algR in the pathogenicity of P. syringae pv.
mental signals such as high osmolarity, starvation for              syringae FF5 by comparing virulence and epiphytic fitness of
nitrogen and/or carbon, heat shock and oxidative stress             wild-type and algR mutant strains.
(Shankar et al., 1995).

The algD gene, which encodes GDP-mannose dehydrogen-                METHODS
ase (Deretic et al., 1987), is the first gene to be transcribed in   Bacterial strains and culture conditions. The bacterial strains
the alginate biosynthetic cluster of both P. aeruginosa and         and plasmids used in this study are listed in Table 1. Pseudomonas
P. syringae (Chitnis & Ohman, 1993; Penaloza-Vazquez      ´         spp. were routinely maintained at 28 uC on King’s medium B (King
et al., 1997). The algC gene, which does not map with algD          et al., 1954), mannitol-glutamate (MG) medium (Keane et al., 1970)
and the other alginate structural genes, encodes phospho-           or MG supplemented with yeast extract at 0?25 g l21 (MGY).
mannomutase, an enzyme that catalyses the second step in            Escherichia coli strains were grown on LB medium or in Terrific
                                                                    Broth (TB) (Sambrook et al., 1989; Miller, 1972) at 37 uC.
alginate biosynthesis by converting mannose 6-phosphate             Antibiotics were added to media at the following concentrations
to mannose 1-phosphate. AlgC is also involved in LPS                (mg ml21): ampicillin (100), tetracycline (25), kanamycin (25),
biosynthesis through its phosphoglucomutase activity,               streptomycin (25) and chloramphenicol (25).
which is required for the synthesis of the complete LPS
core (Coyne et al., 1994). AlgC also participates in                Molecular genetic techniques. Plasmid DNA was isolated from
                                                                    Pseudomonas spp. by alkaline lysis (Sambrook et al., 1989).
rhamnolipid production, presumably by catalysing the con-           Restriction enzyme digests, agarose gel electrophoresis, Southern
version of glucose 6-phosphate to glucose 1-phosphate, the          transfers, colony hybridization and isolation of DNA fragments were
first step in the deoxy-thymidine-diphospho-L-rhamnose               performed using standard protocols (Sambrook et al., 1989).
(dTDP-L-rhamnose) pathway (Olvera et al., 1999). In P.              Plasmid DNA was prepared for DNA sequencing using the Plasmid
aeruginosa, both algD and algC are under positive control of        DNA Midi Kit (Qiagen), and genomic DNA was isolated from P.
AlgR, which functions as a response-regulator member of             syringae using established procedures (Staskawicz et al., 1984).
                                                                    Clones were introduced into recipient strains using a triparental
the two-component signal transduction system and binds to
                                                                    mating procedure (Bender et al., 1991) or by electroporation
multiple sites upstream of algC and algD (Deretic et al.,           (Sambrook et al., 1989).
1989; Kato & Chakrabarty, 1991; Mohr et al., 1992; Zielinski
et al., 1992). P. aeruginosa AlgR was previously over-              DNA fragments were isolated from agarose gels by electroelution
produced, purified, and shown to be required for                     and labelled with digoxigenin (Genius Labelling and Detection Kit;
transcriptional activation of the algD and algC promoter            Roche Molecular Biochemicals) or with [a-32P]dCTP using the Rad
                                                                    Prime DNA Labelling System (Gibco-BRL). Hybridization and post-
regions (Kato & Chakrabarty, 1991; Mohr et al., 1992;               hybridization washes were conducted under high-stringency condi-
Zielinski et al., 1992).                                            tions. A P. syringae pv. syringae FF5 genomic library (Kidambi et al.,
                                                                    1995) was screened for an algC homologue by hybridization with
Strains of P. syringae pv. syringae were previously isolated        pNZ15 (containing algC from P. aeruginosa); hybridization was
from diseased pear trees grown as nursery stock in eastern          conducted for 2 days at 42 uC (Fett et al., 1992).
Oklahoma (Sundin & Bender, 1993). Many of these
P. syringae strains were resistant to copper bactericides           The 0?8 kb XhoI–HindIII DNA fragment containing the algC promoter
(Sundin et al., 1994), and exposure to copper ions                  region of P. syringae pv. syringae FF5 was cloned by PCR amplification
                                                                    using plasmid pMF8.1 as a template (Table 1). Two primers were
stimulated the pathogen to produce copious amounts of               synthesized by the Oklahoma State University (OSU) Recombinant
alginate (Kidambi et al., 1995). This finding prompted us to         DNA/Protein Resource Facility: forward primer, 59-CCCAA-
study the biosynthesis and regulation of alginate production        GCTTCTCGAGTTCACGCCC (XhoI site is underscored), and reverse
in P. syringae pv. syringae FF5, a pathogen associated with a       primer, 59-CCCAAGCTTGCCGTTGTAGTCCTT (HindIII site is
dieback and canker disease of ornamental pear (Sundin &             underscored). The 0?747 kb BamHI–PstI DNA fragment containing
Bender, 1993). Although the biosynthetic genes for alginate         algR from P. syringae pv. syringae FF5 was amplified by PCR using
                                                                    plasmid pMF6.2 as a template (Table 1). Two oligonucleotide primers
production were shown to be conserved between P. syringae
                                                                    were synthesized: forward primer, 59-TGCGGATCCATGAATGT-
                               ˜        ´
FF5 and P. aeruginosa (Penaloza-Vazquez et al., 1997),              CCTGATCGT (BamHI site is underscored), and reverse primer,
aspects of alginate regulation were clearly different in these      59-TACCTGCAGCTAGAGCTGCTGCATCAT (PstI site is underscored).
two pseudomonads (Keith & Bender, 1999, 2001). For
example, in P. syringae FF5, a functional copy of algR was          Glucuronidase (GUS) assays. The PsalgC : : uidA fusion in pMF8.2
                                                                    was introduced into P. syringae pv. syringae FF5, FF5.32 (algR mutant)
not required for algD transcription; however, algR mutants          and FF5.32(pMF6.22). Strains were grown for 24 h on MG agar con-
remained nonmucoid, suggesting an undefined role for algR            taining chloramphenicol, inoculated (OD600 0?1) into MG medium
in the regulation of alginate production in P. syringae (Fakhr      and incubated at 28 uC (250 r.p.m.). Aliquots of cells (three replicates
et al., 1999).                                                      per sampling) were removed at 10 and 20 h after inoculation and ana-
                                                                    lysed for GUS activity as described previously (Palmer et al., 1997).
The present study was undertaken to determine whether               GUS activity was expressed in U (mg protein)21, with 1 U equivalent
AlgR regulates alginate production in P. syringae by                to 1 nmol methylumbelliferone formed per minute. The protein con-
functioning as a positive activator of algC. Transcriptional        tent in cell lysates was determined using the Bio-Rad Protein Assay Kit
                                                                    as recommended by the manufacturer.
fusions and gel retardation studies were utilized to
demonstrate that AlgR functions in the transcriptional              Alginate assays. Selected strains were grown on MG agar (three
activation of algC by binding to its promoter region. We also       plates per strain) supplemented with appropriate antibiotics at 28 uC for

2728                                                                                                                     Microbiology 150
                                                                                                        Role of AlgR in Pseudomonas syringae

Table 1. Bacterial strains and plasmids

 Strain or plasmid                                     Relevant characteristics                                    Reference or source

 E. coli
 DH5a                            D(lacZYA–argF)u169                                                        Sambrook et al. (1989)
 P. syringae pv. syringae
 FF5                             Cur Smr; mucoid; contains pPSR12; originally isolated from                Kidambi et al. (1995)
                                  Bradford pear
 FF5.32                          Cur Kmr; contains pPSR12; nonmucoid; algR : : Tn5                         Fakhr et al. (1999)
 P. syringae pv. glycinea
 PG4180                          Wild-type; pathogenic on soybeans                                         Bender et al. (1993)
 PG4180.K2                       Nonpathogenic; Kmr; rpoN : : nptII                                             ´
                                                                                                           Alarcon-Chaidez et al. (2003)
 pPSR12                          Cur Smr; 200 kb; confers constitutive alginate production to              Kidambi et al. (1995)
                                  P. syringae pv. syringae FF5
 pRK415                          Tcr; RK2-derived cloning vector                                           Keen et al. (1988)
 pRK7813                         Tcr; cosmid vector                                                        Jones & Gutterson (1987)
 pBluescript II SK+              Apr; ColE1 origin; cloning vehicle                                        Stratagene
 pBBR1MCS                        Cmr; 4?7 kb broad-host-range cloning vector                               Kovach et al. (1994)
 pBBR.Gus                        Cmr; 6?6 kb broad-host-range vector containing a promoterless                ˜        ´
                                                                                                           Penaloza-Vazquez & Bender (1998)
                                  uidA gene
 pNZ15                           Kmr; contains 2?6 kb HindIII–SstI fragment with algC from                 Zielinski et al. (1991)
                                  P. aeruginosa in pJRD215
 pMAL-c2                         Apr; ColE1 origin; tac promoter; encodes malE and lacZa                   New England Biolabs
 pMF6.2                          Apr; pBluescript SK+; contains 2?0 kb PstI fragment carrying              Fakhr et al. (1999)
                                  the algR gene from pMF6 cosmid
 pMF6.22                         Tcr; pRK415 with 2?0 kb PstI fragment from pMF6.2 in the                  Fakhr et al. (1999)
                                  transcriptionally inactive orientation with respect to lacZ
 pAP2                            Cmr; pBBR1MCS carrying algR in 2?0 kb PstI fragment;                      This study
                                  derived from pMF6.2
 pMF6.4                          Apr; contains algR as a 0?747 kb BamHI–PstI fragment in                   This study
 pMF8                            Tcr; cosmid clone from P. syringae FF5 in pRK7813                         This study
 pMF8.1                          Apr; contains a 3?9 kb XhoI fragment from pMF8 in pBluescript             This study
 pMF8.2                          Apr; contains a 0?8 kb XhoI–HindIII fragment in pBluescript SK+           This study
 pMF8.3                          Cmr; contains a 0?8 kb XhoI–HindIII fragment of the algC                  This study
                                  promoter in pBBR.Gus in transcriptionally active orientation

72 h. Cells were washed from each plate and resuspended in 0?9 %            Translational fusions. The production of translational fusions
NaCl. Alginate isolation and quantification were performed as described      between the maltose-binding protein (MBP) and AlgR were evaluated
by May & Chakrabarty (1994), and alginic acid from seaweed                  in E. coli DH5a. Cells were grown at 18 uC in TB to an OD600 0?4–0?5,
(Macrocystis pyrifera; Sigma) was used as a standard. The experiment        induced with 1 mM IPTG and incubated for an additional 6 h.
was repeated twice, and means were expressed as the quantity of algi-       Aliquots of cells (1 ml) were removed before and after induction, pel-
nate produced per mg cellular protein. In complementation experi-           leted by centrifugation, resuspended in lysis buffer (Sambrook et al.,
ments, pMF6.22 and pAP2 were introduced into the algR mutant                1989) and incubated on ice for 30 min. The cell suspension was then
FF5.32, and alginate production was assessed as described above.            sonicated as described by Riggs (1994) and centrifuged at 14 000 g for
                                                                            20 min at 4 uC. The pellet was discarded and the supernatant (which
DNA sequencing and analysis. Automated DNA sequencing was                   contains the soluble fraction of the crude extract) was analysed by
provided by the OSU Recombinant DNA/Protein Resource Facility               SDS-PAGE on a 12 % polyacrylamide gel (Sambrook et al., 1989).
and was performed with an ABI 373A apparatus and the ABI
PRISM Dye Primer Cycle Sequencing Kit (Perkin-Elmer).                       Crude cellular lysates containing the MBP–AlgR fusion were isolated
Oligonucleotide primers used for sequencing were also synthesized           from E. coli DH5a(pMF6.4) as described above, except that the cells
by the OSU Recombinant DNA/Protein Resource Facility. Sequence              were grown for 15 h after induction with 1 mM IPTG. Subsequent
manipulations, amino acid alignments and restriction maps were              steps were performed at 0–4 uC in TEDG buffer [50 mM Tris (pH 7?5),
constructed using the Vector NTI Suite, Version 6.0 (Invitrogen).           0?5 mM EDTA, 2 mM DTT, 10 % (v/v) glycerol]. Cells were harvested
Database searches were performed with the BLAST service of the              by centrifugation (500 g, 1 min), supernatants were discarded, and
National Center for Biotechnology Information.                              cells were washed in TEDG buffer and collected by centrifugation. Cells                                                                                                                   2729
A. Penaloza-Vazquez and others

were then resuspended in TEDG buffer and lysed by sonication. Lysates      RESULTS
were centrifuged at 23 000 g for 10 min at 4 uC; supernatants were then
collected and used in gel shift assays.
                                                                           Cloning of the algC promoter from P. syringae
                                                                           A 2?6 kb HindIII–SstI fragment from pNZ15 (containing
Gel retardation experiments. To facilitate end labelling with
[a-32P]dCTP, DNA fragments used for gel retardation were excised           algC and promoter region from P. aeruginosa) was used to
with enzymes that generate 59 overhanging ends. DNA fragments              screen a genomic library of P. syringae pv. syringae FF5 for
were then separated on 5 % polyacrylamide gels and end-labelled            clones containing the P. syringae algC homologue. A cosmid
with [a-32P]dCTP (Sambrook et al., 1989). The concentration of             clone designated pMF8 hybridized with the probe and was
MBP–AlgR used in gel shift assays was evaluated by loading different       chosen for further study. Restriction digestion of pMF8 and
volumes of the soluble protein fraction from E. coli DH5a(pMF6.4)          Southern blot analysis revealed a 3?9 kb XhoI fragment that
to 10 % polyacrylamide gels containing known amounts of bovine
                                                                           hybridized to the probe; this fragment was gel-purified and
serum albumin. Gels were stained with Coomassie blue, and the
concentration of MBP–AlgR was determined using the Bio-Rad GS-
                                                                           cloned in pBluescript II SK+, resulting in pMF8.1. Sequence
700 densitometer and the Molecular Analyst software (version 2.1).         analysis of pMF8.1 showed that the 3?9 kb insert contained
Gel retardation assays were performed by incubating 200 ng MBP–            the coding region of algC along with 456 bp preceding the
AlgR with 2000 c.p.m of end-labelled DNA probe in binding buffer           predicted translational start site. Two PCR primers were
[10mM Tris/HCl (pH 7?5), 10 mM KCl, 1 mM EDTA (pH 8?0),                    designed (Fig. 1, green font, underscored) to amplify the P.
1 mM dithiothreitol, 10 % glycerol and 1 mg poly(dI-dC)]. After            syringae algC promoter region from pMF8.1. The amplified
20 min on ice, 2 ml of loading buffer [binding buffer supplemented         fragment was 810 bp and contained 456 bp upstream of the
with 0?4 % bromophenol blue and 1 % glycerol] was added, and the
samples were loaded onto a 5 % polyacrylamide gel. After electro-
                                                                           translational start site of algC and 354 bp of the predicted
phoresis, gels were dried and autoradiographed.                            coding region. This 0?8 kb fragment was cloned in
                                                                           pBluescript II SK+ and pBBR.Gus as a XhoI–HindIII
                                                                           fragment, resulting in pMF8.2 and pMF8.3, respectively
Pathogenicity and virulence assays. A detached leaf assay                  (Table 1).
(Moragrega et al., 2003; Yessad et al., 1992) was used to evaluate the
pathogenicity of P. syringae pv. syringae FF5 and derivative strains
on Bradford pear (Pyrus calleryana). Bacterial strains were grown for      Sequence analysis of the algC promoter region
48 h on MG agar with antibiotic selection. Bacterial cells were then
suspended in sterile distilled H2O to OD600 0?1 (106 c.f.u. ml21)          CCGTTCGTCN5 was previously reported as a consensus
and used to infiltrate young leaves collected from 2-year-old               sequence recognized and bound by AlgR, and AlgR-binding
Bradford pear trees. Leaves were removed from trees on the day of          sites (ABS) were identified in the promoter regions of algD
inoculation and stored at 4 uC with high relative humidity until
                                                                           and algC from P. aeruginosa (Kato & Chakrabarty, 1991;
inoculation. Prior to inoculation, leaves were surface sterilized in a
solution containing 1 % sodium hypochlorite for 5 min and rinsed           Zielinski et al., 1992). The nucleotide sequence of the region
three times in sterile water. Leaves were then immersed in bacterial       upstream of the translational start site of P. syringae algC
suspensions containing 106 c.f.u. ml21 and vacuum-infiltrated for           contained two putative ABS (Fig. 1, boxed sequences). The
5 min. Leaves were transferred to sterile filter paper and placed on        two binding sites, located at 413 and 75 bp upstream of the
1 % water agar. Petri dishes were sealed with Parafilm and incubated        predicted ATG in P. syringae FF5, resemble algC–ABS1 and
at 26 uC with a 12 h photoperiod. Symptoms were evaluated 10 days          algC–ABS2 in P. aeruginosa (Fujiwara et al., 1993; Zielinski
after inoculation, and five disease severity indices were established.
                                                                           et al., 1992). Within the P. syringae algC coding region, one
Disease severity was ranked from 0 to 4 as follows: 0, no infection;
1, necrotic area ¡2 mm diameter; 2, necrotic area 2–5 mm; 3,               putative ABS was located 45 bp downstream from the
necrosis present in leaf veins and measuring 5–10 mm; 4, necrosis          predicted translational start site, a location that is similar to
exceeding 50 % of the leaf surface area. Disease severity was calcu-       algC–ABS3 in P. aeruginosa (Fujiwara et al., 1993).
lated for each treatment (consisting of five leaves) according to the
formula devised by Moragrega et al. (2003) and is reported as              BLASTN   analysis of the 830 bp sequence shown in Fig. 1
mean±SEM. Statistical significance was assessed by the Duncan               showed 88 % nucleotide identity with the putative algC
multiple range test.                                                       promoter and coding region of P. syringae pv. tomato
                                                                           DC3000 (GenBank AE016856) and 98 % identity with
Epiphytic fitness. The population dynamics of P. syringae pv. syrin-        P. syringae pv. syringae B728a (
gae FF5, FF5.32 (algR mutant) and FF5.32(pAP2) were evaluated by           JGI_microbial/html). The high degree of nucleotide identity
spraying bacterial cells (106 c.f.u. ml21) onto tobacco (Nicotiana taba-   between FF5 and B728 is consistent with their identification
cum cv. Petite Havana) leaves with an airbrush (Keith et al., 2003)        as strains of P. syringae pv. syringae, although their hosts are
until leaf surfaces were uniformly wet. After inoculation, plants were     quite different (pear and bean, respectively). BLASTX analysis
maintained in the greenhouse with a 12 h photoperiod at 20–26 uC.          of the region shown in Fig. 1 indicated 66 % nucleotide
Random leaf samples (one leaf per plant, six leaves total) were
                                                                           identity between P. syringae pv. syringae FF5 and P.
removed at each sampling time (0, 1, 3, 6, 9, 12, 15 and 22 days after
inoculation). Leaves were weighed separately, transferred to 10 ml of      aeruginosa. The algC coding region in P. syringae pv.
sterile 0?01 M potassium phosphate buffer (pH 7?0), and washed for         syringae FF5 showed a high level of amino acid similarity
2 h at 250 r.p.m. Bacterial counts were determined by plating dilu-        (87 %) with phosphomannomutase (AlgC) in P. aeruginosa.
tions onto MG amended with the appropriate antibiotics. Fluorescent
colonies were counted after incubating the plates for 48 h, and the        Sequence analysis of the 2 kb region downstream of algC in
experiment was performed twice.                                            P. syringae pv. syringae FF5 revealed an ORF with 92 %

2730                                                                                                                       Microbiology 150
                                                                                             Role of AlgR in Pseudomonas syringae

                                                                    amino acid similarity to acetylglutamate kinase (ArgB) from
                                                                    P. aeruginosa (GenBank accession no. AE004945) (Fig. 2).
                                                                    Both argB and algC are oriented in the same direction with
                                                                    respect to transcription, and this arrangement is also
                                                                    conserved in P. aeruginosa. The DNA downstream of the
                                                                    argB homologue in P. syringae pv. syringae shows 77 %
                                                                    amino acid similarity to a hypothetical protein in P.
                                                                    aeruginosa (PA5330; GenBank accession no. AE004945).
                                                                    Interestingly, the P. aeruginosa genome contains six genes
                                                                    between argB and the hypothetical protein (PA5330) that
                                                                    are absent in the corresponding regions of P. syringae pv.
                                                                    syringae FF5 and P. syringae pv. tomato DC3000 (Fig. 2).

                                                                    Full expression of the PsalgC promoter requires
                                                                    AlgR and s54
                                                                    In P. aeruginosa, AlgR is required for expression of the algC
                                                                    promoter (PalgC) and binds to PalgC at multiple sites
                                                                    (Zielinski et al., 1991, 1992; Fujiwara et al., 1993). The
                                                                    presence of multiple, putative ABS in the algC promoter
                                                                    region of several P. syringae strains (Fig. 1) suggested that
                                                                    algR might regulate algC expression in this plant pathogen.
                                                                    Furthermore, previous studies conducted by Zielinski et al.
                                                                    (1992) revealed the presence of two s54 recognition motifs
                                                                    upstream of algC in P. aeruginosa, and transcription of
                                                                    algC was significantly reduced in an rpoN mutant of
                                                                    P. aeruginosa. The algC promoter region in P. syringae pv.
                                                                    syringae FF5 contains three sequences resembling s54
                                                                    recognition motifs with the consensus GG-N10-GC
                                                                    (Fig. 1, white font with purple highlighting). This suggests
                                                                    that transcription of algC in P. syringae requires rpoN, which
                                                                    encodes the alternative sigma factor, s54. Although an rpoN
                                                                    mutant of P. syringae FF5 is not available, an rpoN mutant of
                                                                    P. syringae pv. glycinea (Alarcon-Chaidez et al., 2003) was
                                                                    previously constructed and used in this study to assess
                                                                    whether algC transcription is rpoN dependent.

                                                                    To investigate whether algC expression in P. syringae pv.
                                                                    syringae FF5 requires algR and rpoN, the PsalgC promoter
                                                                    was fused to a promoterless uidA gene (pMF8.3, Table 1).
                                                                    Transcriptional activity was evaluated in the following
                                                                    strains carrying pMF8.3: P. syringae pv. syringae FF5 (wild-
                                                                    type), FF5.32 (algR mutant), FF5.32(pMF6.22) (comple-
                                                                    mented algR mutant), P. syringae pv. glycinea PG4180
Fig. 1. Alignment of the algC sequences from P. syringae            (wild-type) and PG4180.K2 (rpoN mutant of PG4180). The
strains FF5, B728a and DC3000, and P. aeruginosa strain
                                                                    vector pBBR.Gus was introduced into all strains, and
PAO1. The P. aeruginosa sequence was previously reported
                                                                    the resulting transformants were used as negative controls.
(Fujiwara et al., 1993; Zielinski et al., 1992); Gaps (--) were
                                                                    The transconjugants from PG4180 and its derivative
used to maximize the alignment. The three reported AlgR bind-
                                                                    PG4180.K2 were grown on minimal medium (MG)
ing sites in P. aeruginosa are indicated in red and boxed. The
three putative AlgR binding sites in P. syringae pv. syringae are
                                                                    supplemented with 0?2 M NaCl. The medium was supple-
shown in black bounded by a rectangle; residues diverging           mented with 0?2 M NaCl because PG4180 is normally
from the consensus AlgR binding site in P. aeruginosa               nonmucoid, and elevated osmolarity is known to stimulate
(CCGTTCGTCN5) are highlighted in yellow. The translational                                                       ˜       ´
                                                                    alginate gene expression in P. syringae (Penaloza-Vazquez
start site (ATG) is highlighted in blue. Sequences that are         et al., 1997). Table 2 shows that algC expression (GUS
homologous to the consensus rpoN recognition sequence, GG-          activity) was reduced approximately fourfold in both FF5.32
N10-GC, are shown in white and highlighted in purple (P. syrin-     and PG4180.K2 as compared with the wild-type FF5 and
gae), and purple or blue (P. aeruginosa). The oligonucleotide       PG4180, respectively, indicating that functional copies of
primers used to amplify the P. syringae algC promoter region        algR and rpoN were required for full activation of algC
are denoted in green and underscored.                               expression in P. syringae.                                                                                                  2731
A. Penaloza-Vazquez and others

                                                                                                                   Fig. 2. Comparison of algC and flanking DNA
                                                                                                                   in P. syringae pv. syringae FF5, P. syringae pv.
                                                                                                                   tomato DC3000 and P. aeruginosa PAO1.
                                                                                                                   P. syringae lacks six ORFs that are present in
                                    algC argB
P. syringae FF5
                                                                                                                   P. aeruginosa PAO1 between argB and the
                                                                                                                   hypothetical protein PA5330. These six ORFs
P. syringae DC3000                                                                                                 (spanned by a bracket) from PA5324 to
                                                       Deleted in P. syringae                                      PA5329 are speculated to encode the follow-
                                                                                                                   ing functions: a probable transcriptional regula-
P. aeruginosa PA01                                                                                                 tor (PA5324), two hypothetical proteins








                                                                                                                   (PA5325, PA5326), a probable oxidoreduc-
                                                                                                                   tase (PA5327), a probable cytochrome c that
                                                                                                                   functions in energy metabolism (PA5328) and
                                                                                                                   a hypothetical unclassified protein (PA5329).
                                                                                                                   Function classes of genes are colour-coded as
                                                                                                                   described by the Pseudomonas Genome Pro-
                                                                                                                   ject (

Overproduction of AlgR from P. syringae                                                          high concentration of AlgR is toxic to E. coli cells,
                                                                                                 which is consistent with previous results in P. aeruginosa
The algR gene was first amplified using PCR and pMF6.2 as
                                                                                                 (Kato & Chakrabarty, 1991). All efforts to purify MBP–AlgR
template DNA (Table 1); restriction sites were incorporated
                                                                                                 using affinity chromatography on amylose resin were
into the oligonucleotide primers to facilitate cloning of a
747 bp BamHI–PstI fragment that contains algR with its
stop codon. This fragment was subcloned into pMAL-c2, a
construct designed for making C-translational fusions to the
                                                                                                 Gel retardation assays
MBP (product of the malE gene). The construct resulting
from this experiment (pMF6.4) was then introduced into                                           The ability of P. syringae AlgR to bind the PsalgC promoter
E. coli DH5a.                                                                                    region was investigated. The 0?8 kb XhoI–HindIII fragment
                                                                                                 in pMF8.2 containing the P. syringae algC promoter was
When E. coli DH5a(pMF6.4) cells were induced with                                                used in gel shift assays. When the 0?8 kb algC promoter
IPTG, a 70 kDa protein was observed (data not shown),                                            fragment was incubated with approximately 200 ng MBP–
which corresponds to the predicted size of the fusion                                            AlgR, migration of the labelled fragment was markedly
protein, MBP–AlgR. This band was absent from uninduced                                           reduced (Fig. 3a, lane 3) as compared with labelled fragment
cells of DH5a(pMF6.4) and from uninduced and induced                                             alone (Fig. 3a, lanes 1 and 4) or labelled fragment incubated
DH5a(pMAL-c2) cells (data not shown). It is important                                            with 200 ng MBP (Fig. 3a, lane 2). These results show that
to note that the MBP–AlgR fusion protein could not be                                            AlgR binds to the algC promoter in P. syringae, possibly
overproduced when E. coli DH5a(pMF6.4) cells were                                                to the ABS sites that are conserved in P. syringae and
induced and incubated at 37 uC; it was necessary to                                              P. aeruginosa. The MBP–AlgR fusion also retarded the
grow cells at 18 uC, a temperature suboptimal for growth,                                        migration of a 0?9 kb XhoI–BssHII fragment from pNZ15,
in order to achieve overproduction. This suggests that a                                         which contains the algC promoter of P. aeruginosa (Fig. 3b,
                                                                                                 lane 3). This is not unexpected given the conservation
                                                                                                 between P. syringae and P. aeruginosa in putative and known
Table 2. Glucuronidase activity [U (mg protein)”1] for                                           ABS; furthermore, algR homologues in the two species are
P. syringae strains containing pMF8.3 (algC–uidA) and                                            highly related (84 % nucleotide identity).
pBBR.Gus (promoterless uidA)
                                                                                                 The specificity of complex formation between MBP–AlgR
Values represent the means from experiments containing three                                     and the fragment containing the P. syringae algC promoter
replicate cultures. Values followed by the same lower-case letter                                was investigated by adding increasing amounts of unlabelled
were not significantly different at P=0?01 using the Duncan mul-                                  XhoI–HindIII fragment to the reaction mixture. When
tiple range test. The experiment was repeated with similar results.                              unlabelled fragment was added as a competitor in amounts
                                                                                                 of 200 ng or higher, binding was either significantly reduced
 Strain                                pMF8.3                            pBBR.Gus
                                                                                                 or completely abolished (Fig. 3c, lanes 6 and 7). However,
 FF5                                   124?75      a
                                                                           7?59c                 when poly(dI-dC) was added to the reaction mixture,
 FF5.32 (algR mutant)                   28?75b                            12?24c                 binding was not altered (Fig. 3c, lanes 3 and 4). These results
 FF5.32(pMF6.22)                       133?15a                            13?41c                 indicate that the MBP–AlgR specifically binds the 0?8 kb
 PG4180                                112?65a                            10?03c                 XhoI–HindIII fragment. This result is consistent with the
 PG4180.K2 (rpoN mutant)                28?18b                             7?65c                 presence of the putative AlgR binding sites in this fragment
                                                                                                 (Fig. 1).

2732                                                                                                                                             Microbiology 150
                                                                                                  Role of AlgR in Pseudomonas syringae

                                                                                      Labelled target DNA
                     (a)                     (b)                        (c)             + MBP-AlgR

                           1   2   3   4           1   2   3     4            1   2    3    4    5     6    7   8

                                                                                  Poly(dl-dC)      Unlabelled
                                                                                                  target DNA

      Fig. 3. Evaluation of AlgR binding to the algC promoter. (a) Gel shift assays of the MBP–AlgR fusion and a 0?8 kb XhoI–
      HindIII fragment containing the algC promoter of P. syringae. Lanes 1 and 4 contain 20 ng end-labelled target DNA and 0 ng
      MBP–AlgR. Lane 2 contains the target DNA fragment and approximately 200 ng MBP. Lane 3 contains the target DNA
      fragment and approximately 200 ng MBP–AlgR. (b) Gel shift assays of the MBP–AlgR fusion and a 0?9 kb XhoI–BssHII
      fragment containing the algC promoter of P. aeruginosa. Lanes 1 and 4 show approximately 20 ng end-labelled target DNA
      and 0 ng MBP–AlgR. Lane 2 contains the target DNA fragment and 200 ng MBP. Lane 3 contains the target DNA fragment
      and 200 ng MBP–AlgR. (c) Competition assays using the MBP–AlgR fusion and the 0?8 kb XhoI–HindIII fragment containing
      the algC promoter from P. syringae. Lanes 1 and 8 show approximately 20 ng end-labelled target DNA and 0 ng MBP–AlgR.
      Lanes 2–7 contain the target DNA fragment and approximately 200 ng MBP–AlgR. The addition of the nonspecific competitor
      poly(dI-dC) is shown in lanes 2–4, which contain 0, 200 and 800 ng poly(dI-dC), respectively. The specific inhibition of
      binding is shown in lanes 5, 6 and 7, which contain 0, 200 and 600 ng unlabelled target fragment.

Complementation studies                                               mean value of 33(±1?3) %. In these leaves, necrosis
                                                                      remained limited to small areas (Fig. 4b). However, leaves
P. syringae pv. syringae FF5 is heavily mucoid when cultured
                                                                      inoculated with the strain FF5.32(pAP2) showed necrosis
on MG or MGY medium (Kidambi et al., 1995). As
expected, the algR mutant FF5.32 remained nonmucoid and               expanding through the main and secondary veins in manner
produced very little alginate (Table 3); however, when                similar to leaves inoculated with the wild-type strain FF5
FF5.32 contained plasmids pMF6.22 and pAP2, both                      (Fig. 4c). The severity value obtained for leaves inoculated
transconjugants were visibly mucoid and produced alginate             with FF5.32(pAP2), 83(±3?3) %, was not significantly
at levels comparable to the parental strain P. syringae pv.           different from that with FF5, indicating that the mutant was
syringae FF5 (Table 3). The two constructs pMF6.22 and
pAP2 both contain algR, but in vectors pRK415 and
pBBR1MCS, respectively. pBBR1MCS, unlike pRK415, is
                                                                      Table 3. Alginate production by derivatives of P. syringae
stable in the absence of antibiotic selection, a property that
                                                                      pv. syringae FF5
was extremely important for plant experiments (see below).
                                                                      Values represent the means from experiments containing three
                                                                      replicate cultures. Values followed by the same lower-case letter
Involvement of algR in pathogenicity and                              were not significantly different at P=0?01 using the Duncan mul-
virulence                                                             tiple range test. The experiment was repeated with similar results.
Detached leaves from Bradford pear (Pyrus calleryana), the
                                                                        Strain                  Alginate production [mg (mg protein)”1]
natural host of P. syringae. pv. syringae FF5, were inoculated
with FF5, FF5.32 and FF5.32(pAP2). Symptoms were                        FF5                                     3100?0a
evaluated 10 days after inoculation, and infection severity             FF5.32                                   401?0b
was recorded. Leaves inoculated with FF5 developed                      FF5.32(pMF6.22)                         2691?0a
maximal disease severity values [92(±3?7) %] and exhibited              FF5.32(pRK415)                           397?0b
necrosis that initiated in the veins and expanded throughout            FF5.32(pAP2)                            2891?0a
the leaf surface (Fig. 4a). Disease severity values obtained            FF5.32(pBBR1MCS)                         388?0b
with FF5.32 (algR mutant) were significantly lower, with a                                                                                                         2733
A. Penaloza-Vazquez and others

                                                                     Fig. 5. Population dynamics of P. syringae pv. syringae strains
                                                                     FF5 (&), FF5.32 (algR mutant; X) and FF5.32(pAP2) (n) on
                                                                     tobacco leaves. Bacterial strains (106 c.f.u. ml”1) were spray-
                                                                     inoculated [approximately 8 p.s.i. (1 p.s.i.=6?9 kPa)] onto
                                                                     tobacco leaves until surfaces were uniformly wet. After inocula-
                                                                     tion, the plants were incubated in the greenhouse with a 12 h
                                                                     photoperiod at 20–26 6C. Bacterial populations were deter-
                                                                     mined by washing the leaves for 2 h at 250 r.p.m. in phosphate
Fig. 4. Bradford pear leaves infiltrated with (a) P. syringae pv.
                                                                     buffer (pH 7?0), followed by dilution plating. All experiments
syringae FF5, (b) FF5.32 (algR mutant), (c) FF5.32(pAP2) and
                                                                     were performed twice with similar results; vertical bars indicate
(d) water. Bacterial inoculum was infiltrated (106 c.f.u. ml”1 at
~94 kPa) into pear leaves by vacuum infiltration. After inocula-
tion, all leaves were incubated in Petri dishes containing mois-
tened filter paper on 1 % (w/v) water agar. Petri dishes were
sealed with Parafilm and incubated at 26 6C with a 12 h photo-        DISCUSSION
period. Photographs were taken 10 days after inoculation.
                                                                     algC is regulated by AlgR and s54 in
                                                                     P. syringae
complemented for pathogenicity by introducing algR in
                                                                     Fujiwara et al. (1993) demonstrated that activation of the
trans. Symptoms were not observed in water-inoculated
                                                                     algC promoter by AlgR in P. aeruginosa was independent
leaves (Fig. 4c).
                                                                     of the relative location (upstream or downstream of the
                                                                     transcriptional start), the number of copies or the
Contribution of algR to epiphytic fitness                             orientation of the ABS. Consequently, the authors of that
The involvement of algR gene in epiphytic fitness was                 study speculated that the ABS may function like a eukaryotic
evaluated by monitoring the epiphytic population of P.               enhancer element and could facilitate the formation of a
syringae pv. syringae FF5, FF5.32 and FF5.32(pAP2) on                DNA loop when AlgR is bound. In P. syringae, three putative
tobacco, which is not a host for P. syringae pv. syringae FF5        ABSs were identified: two upstream of the translational
(Fig. 5). Prior to these experiments, FF5, FF5.32 and                start site and one within the algC coding region. The
FF5.32(pAP2) were evaluated for growth in vitro, and the             gel retardation experiments clearly demonstrated that
results indicated that all three strains grew to similar levels in   P. syringae AlgR bound strongly and specifically to DNA
liquid culture (data not shown). The population of P.                fragments containing the ABS from both P. syringae and
syringae pv. syringae FF5 on day 0 was 1?26106 c.f.u. g21            P. aeruginosa. Therefore, it is highly likely that AlgR from
and remained relatively stable throughout the sampling               P. syringae recognizes the ABS in P. aeruginosa, and that
period (Fig. 5). The population of FF5.32 was approxi-               these binding sequences are conserved in P. syringae. The
mately 2?16106 c.f.u. g21 on day 0; however, this strain             absence of ABS in the algD promoter of P. syringae may
failed to survive on the leaves and the population was               explain why P. syringae, unlike P. aeruginosa, does not
approximately 1000-fold lower than FF5 on day 22 (Fig. 5).           require a functional copy of algR for algD transcriptional
The epiphytic population of FF5.32(pAP2) was lower than              activity (Fakhr et al., 1999).
FF5 during the last 10 days of the sampling period; however,
a significant difference (non-overlapping error bars) was             During the infection process, P. syringae controls gene
only detected on day 22. It is possible that the vector was less     expression via global regulators such as the gacA/gacS
stable during the latter sampling dates. In summary, our             two-component regulatory system (Chatterjee et al.,
results indicate that the supplying algR in trans comple-            2003) and rpoN (Hendrickson et al., 2000; Totten et al.,
mented the epiphytic defects in FF5.32 during the first half          1990). Therefore, cross-talk between the major regulatory
of the experiment, and demonstrate that algR contributes to          systems and transcriptional activators must be coordinated.
the epiphytic survival of P. syringae FF5.                           For example, algC, which functions in the production

2734                                                                                                                 Microbiology 150
                                                                                              Role of AlgR in Pseudomonas syringae

of both LPS and alginate, is transcriptionally activated           The symptoms induced by P. syringae pv. syringae on pear
by AlgR and s54 in P. aeruginosa (Zielinski et al., 1992) and      consist of cankers and necrotic lesions on branches and
P. syringae (this study). However, it is not clear whether         leaves (Yessad et al., 1992). The pathogenicity of P. syringae
AlgR and s54 interact simultaneously to positively activate        pv. syringae is notoriously difficult to assay on deciduous
algC transcription.                                                trees, partly because only young tissues are susceptible and
                                                                   the environmental conditions are critical for proper disease
Possible roles for AlgR in P. syringae                             development in nature (Latorre et al., 2000; Yessad et al.,
                                                                   1992). However, previous results show that detached leaf
Currently, except for algC, other genes regulated by AlgR in       assays are a reliable method for assessing the pathogenicity
P. syringae remain unidentified. Recently, Nikolskaya &             of P. syringae pv. syringae on pear (Moragrega et al., 2003;
Galperin (2002) proposed that AlgR, LytR and AgrR                  Yessad et al., 1992). In the current study, a detached leaf
constitute a new family of transcriptional regulators (the         assay was effective in showing the reduced virulence of the
LytTR or ‘litter’ family), which is based on a conserved           algR mutant FF5.32 (Fig. 4b) on pear leaves, and it was also
DNA-binding domain that is present in the bacterial                used to demonstrate partial complementation of the mutant
genomes recognized by these regulators. These authors              (Fig. 4c). Furthermore, the symptoms observed with wild-
suggest that AlgR may bind to imperfect direct repeats of the      type P. syringae pv. syringae FF5 (Fig. 4a), which include
sequence pattern [TA][AC][CA]GTTN[AG][TG], and may                 lateral spread of necrosis from the main vein, suggest that
possibly bind its target promoters as a dimer. A cursory           this strain can move systemically in leaf tissue. This
examination of the P. syringae pv. tomato DC3000 genome            observation is supported by the elegant microscopy studies
using the PsalgC AlgR-binding sites as search parameters           conducted by Roos & Hattinghe (1987) and Hattingh et al.
revealed 25 ORFs that contain potential ABS. These can be          (1989), which clearly show that P. syringae pv. syringae can
subdivided into the following categories based on homo-            colonize the xylem vessels in leaf veins of woody, deciduous
logy: (i) siderophore biosynthesis, (ii) amino acid biosyn-        host plants. Furthermore, when detached pear leaves were
thesis and metabolism, (iii) carbon catabolism, (iv)               inoculated with P. syringae pv. syringae, Yessad et al. (1992)
chaperones and heat shock factors, (v) polysaccharide              observed necrotic lesions in the veins and lamina within a
synthesis, (vi) phospholipid metabolism, (vii) motility and        few days after inoculation, findings which are consistent
attachment, (viii) nucleotide biosynthesis and metabolism,         with the present study.
(ix) type III effectors, (x) transcriptional regulators and (xi)
transport of small molecules. One of the largest categories        P. syringae pv. syringae overwinters on dormant buds, and
potentially regulated by AlgR is transcription, suggesting         during the growing season, large epiphytic populations of
that this protein may be involved in regulatory cascades.          the pathogen occur on flowers, leaves and other surfaces of
Collectively, these observations suggest that AlgR may have        the growing tree (Manceau et al., 1990; Moragrega et al.,
a broader role in the stress response and pathogenicity of         2003; Yessad et al., 1992). Therefore the epiphytic phase of
P. syringae.                                                       the pathogen life cycle is an important predictor of disease
                                                                   severity. In the present study, we show that the algR mutant,
In P. aeruginosa, Lizewski et al. (2002) compared protein          FF5.32, was impaired relative to the wild-type strain in its
expression in P. aeruginosa and an algR mutant. Their results      ability to colonize a non-host plant, tobacco. This is
indicated that AlgR differentially regulated 47 protein            consistent with an earlier study where an alginate-defective
products. The P. aeruginosa algR mutant was also reduced           mutant of P. syringae pv. syringae 3525 (a bean pathogen)
in virulence as compared to the wild-type in several murine        was impaired in epiphytic fitness (Yu et al., 1999).
models (Lizewski et al., 2002). Inactivation of algR in
P. aeruginosa also eliminated twitching motility, implying         Our results with the algR mutant are especially intriguing, since
that algR has a role in the function of type IV fimbriae            this mutant failed to induce spreading, laminar necrosis in
(Lizewski et al., 2002; Whitchurch et al., 1996). Type IV          detached pear leaves. These results suggest, but do not prove,
pili were required for attachment and biofilm formation             that the algR mutant is impaired in its ability to spread within
in P. aeruginosa (Comolli et al., 1999; O’Toole & Kolter,          the host. However, it is not clear from this study whether the
1998). Although type IV pili have been identified in                defect is primarily due to the lack of alginate production. It is
P. syringae (Roine et al., 1998), our efforts to demonstrate       important to remember that algR has multiple roles in P.
twitching motility in P. syringae FF5 were unsuccessful            aeruginosa (Lizewski et al., 2002; Whitchurch et al., 1996), and
(Fakhr et al., 1999).                                              this is likely to be true in P. syringae. Future studies are planned
                                                                   to compare the pathogenicity and epiphytic fitness of the algR
AlgR functions in the virulence of P. syringae                     mutant with strains defective in structural genes for alginate
pv. syringae                                                       and LPS biosynthesis.

The infection of host plants by P. syringae involves epiphytic
(surface) colonization, entry, establishment of infection sites
in the intercellular spaces (apoplast), multiplication within      ACKNOWLEDGEMENTS
host tissue and production of disease symptoms (Alfano &           This work was supported by grant AI 43311 from the National
Collmer, 1996; Boch et al., 2002; Hirano & Upper, 2000).           Institutes of Health and the Oklahoma Agricultural Experiment                                                                                                       2735
A. Penaloza-Vazquez and others

Station (C. L. B). M. F. acknowledges financial support from the            Hattingh, M. J., Roos, I. M. M. & Mansvelt, E. L. (1989). Infection and
Egyptian government for his dissertation research. We thank Liliana        systemic invasion of deciduous fruit trees by Pseudomonas syringae in
Lopez for technical assistance.                                            South Africa. Plant Dis 73, 784–789.
                                                                                                                ˜        ´
                                                                           Hendrickson, E. L., Guevera, P., Penaloza-Vazquez, A., Shao, J.,
                                                                           Bender, C. & Ausubel, F. M. (2000). Virulence of the phytopathogen
                                                                           Pseudomonas syringae pathovar maculicola is rpoN dependent.
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