1 Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA
2 Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA
3 Departamento de Ingeniería Genética de Plantas CINVESTAV-IPN Unidad Irapuato, Irapuato, Guanajuato, 36500 Mexico
Correspondence
Carol L. Bender
cbender{at}okstate.edu
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AY575079.
Present address: Dept of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58105, USA.
These two authors contributed equally to this work.
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INTRODUCTION |
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The algD gene, which encodes GDP-mannose dehydrogenase (Deretic et al., 1987), is the first gene to be transcribed in the alginate biosynthetic cluster of both P. aeruginosa and P. syringae (Chitnis & Ohman, 1993
; Peñaloza-Vázquez et al., 1997
). The algC gene, which does not map with algD and the other alginate structural genes, encodes phosphomannomutase, an enzyme that catalyses the second step in alginate biosynthesis by converting mannose 6-phosphate to mannose 1-phosphate. AlgC is also involved in LPS biosynthesis through its phosphoglucomutase activity, which is required for the synthesis of the complete LPS core (Coyne et al., 1994
). AlgC also participates in rhamnolipid production, presumably by catalysing the conversion of glucose 6-phosphate to glucose 1-phosphate, the first step in the deoxy-thymidine-diphospho-L-rhamnose (dtdp-L-rhamnose) pathway (Olvera et al., 1999
). In P. aeruginosa, both algD and algC are under positive control of AlgR, which functions as a response-regulator member of the two-component signal transduction system and binds to multiple sites upstream of algC and algD (Deretic et al., 1989
; Kato & Chakrabarty, 1991
; Mohr et al., 1992
; Zielinski et al., 1992
). P. aeruginosa AlgR was previously overproduced, purified, and shown to be required for transcriptional activation of the algD and algC promoter regions (Kato & Chakrabarty, 1991
; Mohr et al., 1992
; Zielinski et al., 1992
).
Strains of P. syringae pv. syringae were previously isolated from diseased pear trees grown as nursery stock in eastern Oklahoma (Sundin & Bender, 1993). Many of these P. syringae strains were resistant to copper bactericides (Sundin et al., 1994
), and exposure to copper ions stimulated the pathogen to produce copious amounts of alginate (Kidambi et al., 1995
). This finding prompted us to study the biosynthesis and regulation of alginate production in P. syringae pv. syringae FF5, a pathogen associated with a dieback and canker disease of ornamental pear (Sundin & Bender, 1993
). Although the biosynthetic genes for alginate production were shown to be conserved between P. syringae FF5 and P. aeruginosa (Peñaloza-Vázquez et al., 1997
), aspects of alginate regulation were clearly different in these two pseudomonads (Keith & Bender, 1999
, 2001
). For example, in P. syringae FF5, a functional copy of algR was not required for algD transcription; however, algR mutants remained nonmucoid, suggesting an undefined role for algR in the regulation of alginate production in P. syringae (Fakhr et al., 1999
).
The present study was undertaken to determine whether AlgR regulates alginate production in P. syringae by functioning as a positive activator of algC. Transcriptional fusions and gel retardation studies were utilized to demonstrate that AlgR functions in the transcriptional activation of algC by binding to its promoter region. We also show a role for algR in the pathogenicity of P. syringae pv. syringae FF5 by comparing virulence and epiphytic fitness of wild-type and algR mutant strains.
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METHODS |
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DNA fragments were isolated from agarose gels by electroelution and labelled with digoxigenin (Genius Labelling and Detection Kit; Roche Molecular Biochemicals) or with [-32P]dCTP using the Rad Prime DNA Labelling System (Gibco-BRL). Hybridization and post-hybridization washes were conducted under high-stringency conditions. A P. syringae pv. syringae FF5 genomic library (Kidambi et al., 1995
) was screened for an algC homologue by hybridization with pNZ15 (containing algC from P. aeruginosa); hybridization was conducted for 2 days at 42 °C (Fett et al., 1992
).
The 0·8 kb XhoIHindIII DNA fragment containing the algC promoter region of P. syringae pv. syringae FF5 was cloned by PCR amplification using plasmid pMF8.1 as a template (Table 1). Two primers were synthesized by the Oklahoma State University (OSU) Recombinant DNA/Protein Resource Facility: forward primer, 5'-CCCAAGCTTCTCGAGTTCACGCCC (XhoI site is underscored), and reverse primer, 5'-CCCAAGCTTGCCGTTGTAGTCCTT (HindIII site is underscored). The 0·747 kb BamHIPstI DNA fragment containing algR from P. syringae pv. syringae FF5 was amplified by PCR using plasmid pMF6.2 as a template (Table 1
). Two oligonucleotide primers were synthesized: forward primer, 5'-TGCGGATCCATGAATGTCCTGATCGT (BamHI site is underscored), and reverse primer, 5'-TACCTGCAGCTAGAGCTGCTGCATCAT (PstI site is underscored).
Glucuronidase (GUS) assays.
The PsalgC : : uidA fusion in pMF8.2 was introduced into P. syringae pv. syringae FF5, FF5.32 (algR mutant) and FF5.32(pMF6.22). Strains were grown for 24 h on MG agar containing chloramphenicol, inoculated (OD600 0·1) into MG medium and incubated at 28 °C (250 r.p.m.). Aliquots of cells (three replicates per sampling) were removed at 10 and 20 h after inoculation and analysed for GUS activity as described previously (Palmer et al., 1997). GUS activity was expressed in U (mg protein)1, with 1 U equivalent to 1 nmol methylumbelliferone formed per minute. The protein content in cell lysates was determined using the Bio-Rad Protein Assay Kit as recommended by the manufacturer.
Alginate assays.
Selected strains were grown on MG agar (three plates per strain) supplemented with appropriate antibiotics at 28 °C for 72 h. Cells were washed from each plate and resuspended in 0·9 % NaCl. Alginate isolation and quantification were performed as described by May & Chakrabarty (1994), and alginic acid from seaweed (Macrocystis pyrifera; Sigma) was used as a standard. The experiment was repeated twice, and means were expressed as the quantity of alginate produced per mg cellular protein. In complementation experiments, pMF6.22 and pAP2 were introduced into the algR mutant FF5.32, and alginate production was assessed as described above.
DNA sequencing and analysis.
Automated DNA sequencing was provided by the OSU Recombinant DNA/Protein Resource Facility and was performed with an ABI 373A apparatus and the ABI PRISM Dye Primer Cycle Sequencing Kit (Perkin-Elmer). Oligonucleotide primers used for sequencing were also synthesized by the OSU Recombinant DNA/Protein Resource Facility. Sequence manipulations, amino acid alignments and restriction maps were constructed using the Vector NTI Suite, Version 6.0 (Invitrogen). Database searches were performed with the BLAST service of the National Center for Biotechnology Information.
Translational fusions.
The production of translational fusions between the maltose-binding protein (MBP) and AlgR were evaluated in E. coli DH5. Cells were grown at 18 °C in TB to an OD600 0·40·5, induced with 1 mM IPTG and incubated for an additional 6 h. Aliquots of cells (1 ml) were removed before and after induction, pelleted by centrifugation, resuspended in lysis buffer (Sambrook et al., 1989
) and incubated on ice for 30 min. The cell suspension was then sonicated as described by Riggs (1994)
and centrifuged at 14 000 g for 20 min at 4 °C. The pellet was discarded and the supernatant (which contains the soluble fraction of the crude extract) was analysed by SDS-PAGE on a 12 % polyacrylamide gel (Sambrook et al., 1989
).
Crude cellular lysates containing the MBPAlgR fusion were isolated from E. coli DH5(pMF6.4) as described above, except that the cells were grown for 15 h after induction with 1 mM IPTG. Subsequent steps were performed at 04 °C in TEDG buffer [50 mM Tris (pH 7·5), 0·5 mM EDTA, 2 mM DTT, 10 % (v/v) glycerol]. Cells were harvested by centrifugation (500 g, 1 min), supernatants were discarded, and cells were washed in TEDG buffer and collected by centrifugation. Cells were then resuspended in TEDG buffer and lysed by sonication. Lysates were centrifuged at 23 000 g for 10 min at 4 °C; supernatants were then collected and used in gel shift assays.
Gel retardation experiments.
To facilitate end labelling with [-32P]dCTP, DNA fragments used for gel retardation were excised with enzymes that generate 5' overhanging ends. DNA fragments were then separated on 5 % polyacrylamide gels and end-labelled with [
-32P]dCTP (Sambrook et al., 1989
). The concentration of MBPAlgR used in gel shift assays was evaluated by loading different volumes of the soluble protein fraction from E. coli DH5
(pMF6.4) to 10 % polyacrylamide gels containing known amounts of bovine serum albumin. Gels were stained with Coomassie blue, and the concentration of MBPAlgR was determined using the Bio-Rad GS-700 densitometer and the Molecular Analyst software (version 2.1). Gel retardation assays were performed by incubating 200 ng MBPAlgR with 2000 c.p.m of end-labelled DNA probe in binding buffer [10mM Tris/HCl (pH 7·5), 10 mM KCl, 1 mM EDTA (pH 8·0), 1 mM dithiothreitol, 10 % glycerol and 1 µg poly(dI-dC)]. After 20 min on ice, 2 µl of loading buffer [binding buffer supplemented with 0·4 % bromophenol blue and 1 % glycerol] was added, and the samples were loaded onto a 5 % polyacrylamide gel. After electrophoresis, gels were dried and autoradiographed.
Pathogenicity and virulence assays.
A detached leaf assay (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 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. ml1) and used to infiltrate young leaves collected from 2-year-old Bradford pear trees. Leaves were removed from trees on the day of inoculation and stored at 4 °C with high relative humidity until inoculation. Prior to inoculation, leaves were surface sterilized in a solution containing 1 % sodium hypochlorite for 5 min and rinsed three times in sterile water. Leaves were then immersed in bacterial suspensions containing 106 c.f.u. ml1 and vacuum-infiltrated for 5 min. Leaves were transferred to sterile filter paper and placed on 1 % water agar. Petri dishes were sealed with Parafilm and incubated at 26 °C with a 12 h photoperiod. Symptoms were evaluated 10 days after inoculation, and five disease severity indices were established. Disease severity was ranked from 0 to 4 as follows: 0, no infection; 1, necrotic area
2 mm diameter; 2, necrotic area 25 mm; 3, necrosis present in leaf veins and measuring 510 mm; 4, necrosis exceeding 50 % of the leaf surface area. Disease severity was calculated for each treatment (consisting of five leaves) according to the formula devised by Moragrega et al. (2003)
and is reported as mean±SEM. Statistical significance was assessed by the Duncan multiple range test.
Epiphytic fitness.
The population dynamics of P. syringae pv. syringae FF5, FF5.32 (algR mutant) and FF5.32(pAP2) were evaluated by spraying bacterial cells (106 c.f.u. ml1) onto tobacco (Nicotiana tabacum cv. Petite Havana) leaves with an airbrush (Keith et al., 2003) until leaf surfaces were uniformly wet. After inoculation, plants were maintained in the greenhouse with a 12 h photoperiod at 2026 °C. Random leaf samples (one leaf per plant, six leaves total) were 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 sterile 0·01 M potassium phosphate buffer (pH 7·0), and washed for 2 h at 250 r.p.m. Bacterial counts were determined by plating dilutions onto MG amended with the appropriate antibiotics. Fluorescent colonies were counted after incubating the plates for 48 h, and the experiment was performed twice.
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RESULTS |
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BLASTN analysis of the 830 bp sequence shown in Fig. 1 showed 88 % nucleotide identity with the putative algC promoter and coding region of P. syringae pv. tomato DC3000 (GenBank AE016856) and 98 % identity with P. syringae pv. syringae B728a (http://www.jgi.doe.gov/JGI_microbial/html). The high degree of nucleotide identity between FF5 and B728 is consistent with their identification as strains of P. syringae pv. syringae, although their hosts are quite different (pear and bean, respectively). BLASTX analysis of the region shown in Fig. 1
indicated 66 % nucleotide identity between P. syringae pv. syringae FF5 and P. aeruginosa. The algC coding region in P. syringae pv. syringae FF5 showed a high level of amino acid similarity (87 %) with phosphomannomutase (AlgC) in P. aeruginosa.
Sequence analysis of the 2 kb region downstream of algC in P. syringae pv. syringae FF5 revealed an ORF with 92 % 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
).
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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) (complemented algR mutant), P. syringae pv. glycinea PG4180 (wild-type) and PG4180.K2 (rpoN mutant of PG4180). The vector pBBR.Gus was introduced into all strains, and the resulting transformants were used as negative controls. The transconjugants from PG4180 and its derivative PG4180.K2 were grown on minimal medium (MG) supplemented with 0·2 M NaCl. The medium was supplemented with 0·2 M NaCl because PG4180 is normally nonmucoid, and elevated osmolarity is known to stimulate alginate gene expression in P. syringae (Peñaloza-Vázquez et al., 1997
). Table 2
shows that algC expression (GUS activity) was reduced approximately fourfold in both FF5.32 and PG4180.K2 as compared with the wild-type FF5 and PG4180, respectively, indicating that functional copies of algR and rpoN were required for full activation of algC expression in P. syringae.
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When E. coli DH5(pMF6.4) cells were induced with IPTG, a 70 kDa protein was observed (data not shown), which corresponds to the predicted size of the fusion protein, MBPAlgR. This band was absent from uninduced cells of DH5
(pMF6.4) and from uninduced and induced DH5
(pMAL-c2) cells (data not shown). It is important to note that the MBPAlgR fusion protein could not be overproduced when E. coli DH5
(pMF6.4) cells were induced and incubated at 37 °C; it was necessary to grow cells at 18 °C, a temperature suboptimal for growth, in order to achieve overproduction. This suggests that a high concentration of AlgR is toxic to E. coli cells, which is consistent with previous results in P. aeruginosa (Kato & Chakrabarty, 1991
). All efforts to purify MBPAlgR using affinity chromatography on amylose resin were unsuccessful.
Gel retardation assays
The ability of P. syringae AlgR to bind the PsalgC promoter region was investigated. The 0·8 kb XhoIHindIII fragment in pMF8.2 containing the P. syringae algC promoter was used in gel shift assays. When the 0·8 kb algC promoter fragment was incubated with approximately 200 ng MBPAlgR, migration of the labelled fragment was markedly reduced (Fig. 3a, lane 3) as compared with labelled fragment alone (Fig. 3a
, lanes 1 and 4) or labelled fragment incubated with 200 ng MBP (Fig. 3a
, lane 2). These results show that AlgR binds to the algC promoter in P. syringae, possibly to the ABS sites that are conserved in P. syringae and P. aeruginosa. The MBPAlgR fusion also retarded the migration of a 0·9 kb XhoIBssHII fragment from pNZ15, 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 ABS; furthermore, algR homologues in the two species are highly related (84 % nucleotide identity).
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Complementation studies
P. syringae pv. syringae FF5 is heavily mucoid when cultured on MG or MGY medium (Kidambi et al., 1995). As expected, the algR mutant FF5.32 remained nonmucoid and produced very little alginate (Table 3
); however, when FF5.32 contained plasmids pMF6.22 and pAP2, both transconjugants were visibly mucoid and produced alginate at levels comparable to the parental strain P. syringae pv. 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 stable in the absence of antibiotic selection, a property that was extremely important for plant experiments (see below).
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DISCUSSION |
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During the infection process, P. syringae controls gene expression via global regulators such as the gacA/gacS two-component regulatory system (Chatterjee et al., 2003) and rpoN (Hendrickson et al., 2000
; Totten et al., 1990
). Therefore, cross-talk between the major regulatory systems and transcriptional activators must be coordinated. For example, algC, which functions in the production of both LPS and alginate, is transcriptionally activated by AlgR and
54 in P. aeruginosa (Zielinski et al., 1992
) and P. syringae (this study). However, it is not clear whether AlgR and
54 interact simultaneously to positively activate algC transcription.
Possible roles for AlgR in P. syringae
Currently, except for algC, other genes regulated by AlgR in P. syringae remain unidentified. Recently, Nikolskaya & Galperin (2002) proposed that AlgR, LytR and AgrR constitute a new family of transcriptional regulators (the LytTR or litter family), which is based on a conserved DNA-binding domain that is present in the bacterial genomes recognized by these regulators. These authors suggest that AlgR may bind to imperfect direct repeats of the sequence pattern [TA][AC][CA]GTTN[AG][TG], and may possibly bind its target promoters as a dimer. A cursory examination of the P. syringae pv. tomato DC3000 genome using the PsalgC AlgR-binding sites as search parameters revealed 25 ORFs that contain potential ABS. These can be subdivided into the following categories based on homology: (i) siderophore biosynthesis, (ii) amino acid biosynthesis and metabolism, (iii) carbon catabolism, (iv) chaperones and heat shock factors, (v) polysaccharide synthesis, (vi) phospholipid metabolism, (vii) motility and attachment, (viii) nucleotide biosynthesis and metabolism, (ix) type III effectors, (x) transcriptional regulators and (xi) transport of small molecules. One of the largest categories potentially regulated by AlgR is transcription, suggesting that this protein may be involved in regulatory cascades. Collectively, these observations suggest that AlgR may have a broader role in the stress response and pathogenicity of P. syringae.
In P. aeruginosa, Lizewski et al. (2002) compared protein expression in P. aeruginosa and an algR mutant. Their results indicated that AlgR differentially regulated 47 protein products. The P. aeruginosa algR mutant was also reduced in virulence as compared to the wild-type in several murine models (Lizewski et al., 2002
). Inactivation of algR in P. aeruginosa also eliminated twitching motility, implying that algR has a role in the function of type IV fimbriae (Lizewski et al., 2002
; Whitchurch et al., 1996
). Type IV pili were required for attachment and biofilm formation in P. aeruginosa (Comolli et al., 1999
; O'Toole & Kolter, 1998
). Although type IV pili have been identified in P. syringae (Roine et al., 1998
), our efforts to demonstrate twitching motility in P. syringae FF5 were unsuccessful (Fakhr et al., 1999
).
AlgR functions in the virulence of P. syringae pv. syringae
The infection of host plants by P. syringae involves epiphytic (surface) colonization, entry, establishment of infection sites in the intercellular spaces (apoplast), multiplication within host tissue and production of disease symptoms (Alfano & Collmer, 1996; Boch et al., 2002
; Hirano & Upper, 2000
). The symptoms induced by P. syringae pv. syringae on pear consist of cankers and necrotic lesions on branches and leaves (Yessad et al., 1992
). The pathogenicity of P. syringae pv. syringae is notoriously difficult to assay on deciduous trees, partly because only young tissues are susceptible and the environmental conditions are critical for proper disease development in nature (Latorre et al., 2000
; Yessad et al., 1992
). However, previous results show that detached leaf assays are a reliable method for assessing the pathogenicity of P. syringae pv. syringae on pear (Moragrega et al., 2003
; Yessad et al., 1992
). In the current study, a detached leaf assay was effective in showing the reduced virulence of the algR mutant FF5.32 (Fig. 4b
) on pear leaves, and it was also used to demonstrate partial complementation of the mutant (Fig. 4c
). Furthermore, the symptoms observed with wild-type P. syringae pv. syringae FF5 (Fig. 4a
), which include lateral spread of necrosis from the main vein, suggest that this strain can move systemically in leaf tissue. This observation is supported by the elegant microscopy studies conducted by Roos & Hattinghe (1987)
and Hattingh et al. (1989)
, which clearly show that P. syringae pv. syringae can colonize the xylem vessels in leaf veins of woody, deciduous host plants. Furthermore, when detached pear leaves were inoculated with P. syringae pv. syringae, Yessad et al. (1992)
observed necrotic lesions in the veins and lamina within a few days after inoculation, findings which are consistent with the present study.
P. syringae pv. syringae overwinters on dormant buds, and during the growing season, large epiphytic populations of the pathogen occur on flowers, leaves and other surfaces of the growing tree (Manceau et al., 1990; Moragrega et al., 2003
; Yessad et al., 1992
). Therefore the epiphytic phase of the pathogen life cycle is an important predictor of disease severity. In the present study, we show that the algR mutant, FF5.32, was impaired relative to the wild-type strain in its ability to colonize a non-host plant, tobacco. This is consistent with an earlier study where an alginate-defective mutant of P. syringae pv. syringae 3525 (a bean pathogen) was impaired in epiphytic fitness (Yu et al., 1999
).
Our results with the algR mutant are especially intriguing, since this mutant failed to induce spreading, laminar necrosis in detached pear leaves. These results suggest, but do not prove, that the algR mutant is impaired in its ability to spread within the host. However, it is not clear from this study whether the defect is primarily due to the lack of alginate production. It is important to remember that algR has multiple roles in P. aeruginosa (Lizewski et al., 2002; Whitchurch et al., 1996
), and this is likely to be true in P. syringae. Future studies are planned to compare the pathogenicity and epiphytic fitness of the algR mutant with strains defective in structural genes for alginate and LPS biosynthesis.
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ACKNOWLEDGEMENTS |
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Received 30 March 2004;
revised 16 May 2004;
accepted 18 May 2004.
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