Max-Planck-Institut für Zellbiologie, Rosenhof, 68526 Ladenburg, Germany1
GBF, Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, 38124 Braunschweig, Germany2
Author for correspondence: Klaus Geider. Tel: +49 6203 106 117. Fax: +49 6203 106 122. e-mail: kgeider{at}zellbio.mpg.de
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ABSTRACT |
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Keywords: Asian pear blight, fire blight, amylovoran, sugar linkage, cps genes
Abbreviations: CPC, cetylpyridinium chloride; EPS, exopolysaccharide; HR, hypersensitive reaction
The GenBank accession number for the sequence reported in this paper is AJ300463.
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INTRODUCTION |
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The production of capsular EPS is a common feature of many Gram-negative bacteria (Sutherland, 1988 ). For plant pathogens, EPS capsules and slime are important for protection of the pathogens against recognition by plant defence mechanisms, to bind water, keeping the bacteria moist, and for retention of nutrients and ions released from the damaged plant cells (Leigh & Coplin, 1992
). E. amylovora produces the acidic EPS amylovoran, and the gene cluster encoding amylovoran biosynthesis (ams) was cloned and sequenced (Bugert & Geider, 1995
). Mutation analysis of the synthesis genes showed EPS to be an important virulence factor for the pathogen (Steinberger & Beer, 1988
; Bellemann & Geider, 1992
; Bernhard et al., 1993
). Erwinia stewartii (Pantoea stewartii subsp. stewartii) produces stewartan, and EPS-deficient mutants of E. stewartii failed to cause wilt and water-soaking on corn (Dolph et al., 1988
). Amylovoran and stewartan consist of highly polymerized repeating units (Jumel et al., 1997
). The sugar backbone of a repeating unit of amylovoran comprises only galactose residues (Nimtz et al., 1996a
), whereas in stewartan, glucose substitutes a galactose residue of amylovoran (Nimtz et al., 1996b
). As will be discussed in Results, the major side chain of amylovoran consists of a glucuronic acid residue attached to the backbone of galactose and is terminated by a galactose carrying pyruvyl and acetyl residues. The similar side chain of stewartan is terminated by a glucose residue and carries a further glucose residue as a second side chain of the repeating units. For amylovoran, the number of the repeating units carrying this glucose residue is dependent on the growth conditions of the bacteria, as preparations from suspension cultures showed a low incidence (approx. 10%) of the monosaccharide residue (Nimtz et al., 1996a
), whereas more than half of the repeating units are branched in amylovoran from agar-grown cells.
E. pyrifoliae produces EPS in minimal medium, ooze on slices of immature pears and mucoid colonies on MM2 agar similar to E. amylovora (Rhim et al., 1999 ). In this study, we have investigated the various biochemical properties of the EPS, analysed genes for its synthesis and assessed its importance for virulence.
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METHODS |
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For isolation, EPS was prepared from E. pyrifoliae strain Ep1/96 grown on a cellophane disk, which was placed on MM2A agar (Kim & Geider, 2000 ), for 3 days at 28 °C. The slimy cells were suspended in 3 ml water, vortexed and centrifuged. The supernatant was freeze-dried for storage of the EPS. Amylovoran from strain Ea1/79 was prepared by the same method.
For degradation, EPS (100 µg ml-1) in 100 mM NaCl/10 mM sodium acetate (pH 4·7) was incubated with 175 ng EPS depolymerase from E. amylovora phage Ea1h (Kim & Geider, 2000
) per ml assay mixture at 28 °C. The remaining high-molecular-mass EPS fraction was measured by the CPC method (Bellemann et al., 1994
) every 30 min.
Lectin staining of capsular EPS.
Ep1/96 or mutants were grown for 2 days on MM2A agar and cells from a colony were transferred onto a glass slide. They were suspended in 5 µl FITC-labelled lectin from Abrus precatorius (Sigma), which specifically binds to the galactose residue of the amylovoran side chain. The cells were visualized in the fluorescence microscope Zeiss Axiovert 405 in bright field and UV with an oil immersion lens at 1000-fold total magnification and the filter combination BP450490/FT510/LP520 (excitation filter/dichroic/emission filter) as described previously (Bellemann et al., 1994 ).
Structural characterization of the capsular polysaccharides by methylation analysis, ESI-MS and NMR.
The monosaccharide composition of the native polysaccharide was analysed and the linkage arrangement investigated by methylation analysis as described previously (Nimtz et al., 1996a ).
For mass spectra, the repeating units, obtained by enzymic depolymerization of EPS, were dissolved in methanol/water (50/50, v/v) at a concentration of approximately 5 pmol µl-1, and 3 µl of the solution was introduced into gold-coated nanospray glass capillaries. The tip of the capillary was placed orthogonally in front of the entrance hole of a QTof II mass spectrometer (Micromass) equipped with a nanospray ion source, and a voltage of -800 V was applied. For collision-induced dissociation experiments, parent ions were selectively transmitted from the quadrupole mass analyser into the collision cell. Argon was used as the collision gas and the kinetic energy was set at approximately +25 eV. The resulting daughter ions then were separated by an orthogonal time-of-flight mass analyser.
For 1H NMR spectroscopic analysis, the oligosaccharides were repeatedly lyophilized against D2O (Fluka, >99·95 atom% D) at pD 7 and ambient temperature. 1H NMR spectra at 600 MHz were recorded at 300 K on a Bruker AVANCE DMX 600 NMR spectrometer incorporating a gradient unit. A 1·3 s presaturation pulse was employed prior to the experimental pulse sequence in order to suppress the signal of the residual HOD resonance. All 1D and 2D COSY 1H spectra were recorded using standard Bruker software.
Cloning, sequencing and sequence analysis.
Several primer pairs (1720 oligonucleotides) from E. amylovora used for sequencing of the ams region (Bugert et al., 1995 ) were applied to PCR reactions with E. pyrifoliae (Table 2
). PCR products were separated on a 0·8% agarose gel and DNA fragments of the size expected according to the corresponding ams genes were eluted with a QIAEXII gel extraction kit (QIAGEN). For sequencing analysis, the PCR fragments were cloned into the vector pGEM-T (Promega) and sequenced with an automatic sequencer (ALFexpress; Amersham Pharmacia Biotech). The data were analysed with the sequence analysis programs Clone manager v.5/Align Plus 4 (Scientific & Educational Software). Database searches were performed on the internet with the BLASTP+BEAUTY program (Worley et al., 1995
). The nucleotide sequence data were deposited in the EMBL Nucleotide Sequence Database (accession number AJ300463).
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Pathogenicity assays on immature pear slices or pear seedlings.
Immature pear fruits were harvested in early summer and stored at 4 °C. Pear slices were inoculated with freshly grown bacteria from agar with a toothpick and incubated in an air-sealed Petri dish for at least 6 days at 28 °C before the symptoms were assessed.
In order to grow seedlings from European pears (Pyrus communis cv. Kirchensaller Mostbirne) and from Asian pears (Pyrus pyrifolia cv. Nashi), seeds were vernalized for 5 days in ice water at 4 °C and kept in sand at 10 °C for 24 weeks. Germinated seedlings were potted and incubated in light for 45 weeks at 22 °C. For virulence assays, secondary leaves of the plants were cut at the tip and inoculated with a pipette tip dipped in a suspension of bacteria. Symptoms were monitored after further incubation of the seedlings for 23 weeks.
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RESULTS |
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Alignment of the amino acid sequences deduced from cpsG, H, I, A, B, C with the program BLASTP+BEAUTY (Worley et al., 1995 ) showed a high homology for proteins encoded by the corresponding E. amylovora ams genes. CpsG has 95% identity with AmsG. More than 90% identity was also found for CpsH/AmsH, CpsI/AmsI and CpsB/AmsB. The amino acid sequence of the analysed part of CpsC is 94% identical with the corresponding part of AmsC of E. amylovora.
Construction, virulence and complementation of an E. pyrifoliae EPS mutant
To verify that EPS is also a virulence factor of E. pyrifoliae, the chromosomal cpsB gene was mutated by disruption with a 0·7 kb internal DNA fragment from cpsB. After transformation of Ep1/96 with pfdB14Z'-B1, 120 colonies were picked from StI agar with Cm onto MM2A agar and seven non-mucoid colonies were obtained. Cointegrate formation of pfdB14Z'-B1 with cpsB was confirmed by PCR analysis for these mutants (data not shown). One mutant, Ep1/96-mB1, was assayed for EPS production with CPC. The growth rate of the mutant and the wild-type strain Ep1/96 was identical, but no EPS was detected, similar to the E. amylovora EPS mutant Ea1/79-D32, which is deficient in amsB (Fig. 6). In the lectin staining assay, no EPS capsules were observed for Ep1/96-mB1. Consequently, cpsB is required for EPS synthesis by E. pyrifoliae.
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DISCUSSION |
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For EPS of E. pyrifoliae, the molecular mass of the cleavage products showed the complete absence of a glucose residue linked to O-6 of the central galactose residue as found for repeating units of amylovoran. The data with ESI/MS were confirmed by methylation analysis and 1D and 2D 1H NMR spectroscopy. A higher proportion of acetylation at the terminal pyruvylated galactose of the side branch of E. pyrifoliae was found as a minor difference compared to the E. amylovora EPS originally analysed by us (Nimtz et al., 1996a ). Interestingly, in the amylovoran repeating unit prepared from cells grown on minimal agar, there was a higher degree of acetylation than in the case of amylovoran from suspension cells and additionally about 10% of the acetyl groups were found to be exchanged for succinyl residues, which were not detected in EPS from E. pyrifoliae nor from E. amylovora grown as suspension cells. However, all the acyl substituents were found to be linked exclusively to the terminal galactose residue of the sugar side-chain. Moreover, whereas in EPS preparations from suspension cells only 10% of an additional glucose residue linked to O-6 of the core galactose of the repeating unit was found (Nimtz et al., 1996a
), in the amylovoran preparation from agar-grown cells, approximately 65% of the repeating unit carried this moiety (Figs 2
, 3
and 4
). Therefore, the occurrence of this additional glucose residue and the nature of the acyl substituents in the sugar side-chain are apparently also dependent on the culture conditions used for the growth of the bacteria.
The high degree of similarity of the chemical structures of the two EPS species may not justify naming E. pyrifoliae EPS pyrifolan by analogy to amylovoran. Their relatedness was also evident from the homology between ams and cps genes encoding proteins for biosynthesis of EPS. The corresponding genes were more than 90% homologous to each other. CpsG is assumed to transfer UDP-galactose to the lipid carrier (C. Langlotz & K. Geider, unpublished). CpsA has 92% identity with AmsA, which is a tyrosine kinase associated with production of EPS and virulence of E. amylovora (Ilan et al., 1999 ). CpsB is assumed to be a glycosyl transferase, which transfers galactose from UDP-galactose to galactose attached to the lipid-carrier. AmsI is an acid phosphatase (Bugert & Geider, 1997
). AmsH may be involved in transport of the repeating unit (Bugert & Geider, 1995
) (Table 4
, Figs 2
and 5
). The cps mutant Ep1/96mB1 was successfully complemented with the plasmid pEA109 carrying the intact amsB gene. The gene products CpsB and AmsB are therefore exchangeable for synthesis of the repeating units. On the other hand, the glucose residue at the branched galactose was not observed in the repeating units obtained from the pEA109-complemented mutant (M. Schollmeyer, M. Nimtz & K. Geider, unpublished). It can be concluded that the ams genes on pEA109 do not encode for the responsible sugar transferase. In addition to some differences in the acetylation pattern and substitution by succinyl groups, this glucose residue is a major difference for EPS of E. pyrifoliae and amylovoran of E. amylovora. Amylovoran produced in E. stewartii cps mutants carrying pEA109 is not acetylated, in contrast to stewartan synthesized in E. amylovora ams mutants with pES2144, a cosmid with the cps genes of Pantoea (Erwinia) stewartii (Bernhard et al., 1996
). Genes for acetylation should reside apart from the ams region inserted in plasmid pEA109.
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Received 5 July 2002;
revised 9 September 2002;
accepted 12 September 2002.
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