Expression of bacteriophage {phi}Ea1h lysozyme in Escherichia coli and its activity in growth inhibition of Erwinia amylovora

Won-Sik Kim, Heike Salm and Klaus Geider{dagger}

Max-Planck-Institut für Zellbiologie, Ladenburg, Germany

Correspondence
Klaus Geider
k.geider{at}bba.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A 3·3 kb fragment from Erwinia amylovora phage {phi}Ea1h in plasmid pJH94 was previously characterized and found to contain an exopolysaccharide depolymerase (dpo) gene and two additional ORFs encoding 178 and 119 amino acids. ORF178 (lyz) and ORF119 (hol) were found to overlap by 19 bp and they resembled genes encoding lysozymes and holins. In nucleotide sequence alignments, lyz had structurally conserved regions with residues important for lysozyme function. The lyz gene was cloned into an expression vector and expressed in Escherichia coli. Active lysozyme was detected only when E. coli cells with the lyz gene and a kanamycin-resistance cassette were grown in the presence of kanamycin. Growth of Erw. amylovora was inhibited after addition of enzyme exceeding a threshold for lysozyme to target cells. When immature pears were soaked in lysates of induced cells, symptoms such as ooze formation and necrosis were retarded or inhibited after inoculation with Erw. amylovora.


{dagger}Present address: Max-Planck-Institut für Zellbiologie, c/o Biologische Bundesanstalt, Schwabenheimer Str. 101, 69221 Dossenheim, Germany.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fire blight is a serious bacterial disease in pome fruit orchards; it is endemic in north-eastern America (Jock & Geider, 2004) and in the last century it spread to many countries of the northern hemisphere and to New Zealand (Bonn & van der Zwet, 2000). The causative agent Erwinia amylovora is often transmitted by insects (Hildebrand et al., 2000), especially by bees when visiting flowers. To control fire blight, streptomycin and other antibiotics have been applied successfully (Psallidas & Tsiantos, 2000); however their use has been restricted in many countries. Bacteriophages have been used for identification of plant-associated bacteria, to determine capsulation of Erw. amylovora cells and also for disease control (Billing, 1960; Bernhard et al., 1993; Schnabel & Jones, 2001); they have been isolated from the soil in the vicinity of diseased trees (Ritchie & Klos, 1977) and from diseased tissue of host plants (Okabe & Goto, 1963). A bacteriophage was applied to pear slices and it delayed symptom development after inoculation with Erw. amylovora (Erskine, 1973). Another potentially specific approach for interfering with growth of Erw. amylovora has been the proposed use of Serracin P, a phage-tail-like bacteriocin (Jabrane et al., 2002). In orchards, successful control of fire blight with bacteriophages or bacteriocins has not yet been reported.

In a late stage of their life cycle, many bacteriophages express lysozyme and holin to lyse the host cells (Young, 1992). The holin forms a pore in the host cell membrane to channel the lysozyme into the periplasm. The muramidase activity of lysozyme hydrolyses 1,4-{beta}-linkages between N-acetyl-D-glucosamine and N-acetylmuramic acid in the peptidoglycan layer of bacterial cell walls (Cooper, 1997). Lysozymes are classified into four families: chicken-, goose-, phage- and bacterial-type (Jolles & Jolles, 1984). The amino acid sequences within a family are related, but there are no clear sequence homologies among families (Weaver et al., 1985).

In this study, we investigated the requirements for expression of the lysozyme gene cloned from Erw. amylovora phage {phi}Ea1h into Escherichia coli, and its ability to inhibit growth of Erw. amylovora in broth cultures and on agar plates. These features of lysozyme could be used for control of fire blight in orchards.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and plasmids.
The bacterial strains and plasmids used in this study are listed in Table 1. Media and growth conditions have been described previously (Kim & Geider, 2000). Antibiotics were ampicillin (Ap, 100 µg ml–1), chloramphenicol (Cm, 20 µg ml–1), kanamycin (Km, 20 µg ml–1) and streptomycin (Sm, 500 µg ml–1).


View this table:
[in this window]
[in a new window]
 
Table 1. Bacterial strains and plasmids

 
Cloning of the {phi}Ea1h lysozyme gene.
Primer pairs were designed from the insert in plasmid pJH94 (GenBank accession no. AJ278614) to amplify the lyz gene without the ATG start codon for fusion into a His-tagged expression vector. The forward primer Lyz5 (5'-GCGCGGGATCCTCTGTGAAGAAGGCTCT; a created BamHI site is underlined) and the reverse primer Lyz3c (5'-GCGCGAAGCTTCTACATATATTTTACGC; a created HindIII site is underlined) were applied with 2·5 U Pfu DNA polymerase (Stratagene) as described previously (Kim & Geider, 2000). The amplified fragments were digested with BamHI and HindIII, purified in an agarose gel and eluted with a gel extraction kit (QIAEX II; Qiagen). The 0·5 kb (lyz) PCR fragment was fused to the nucleotide sequence encoding the His tag in plasmid pQE-30, which was useful for detecting the protein with anti-His antibodies. The fusion was confirmed by sequence analysis, and the plasmid was transformed into E. coli strain M15(pREP4).

Preparation of cell lysates with soluble lysozyme.
To express the cloned lysozyme gene in E. coli M15(pREP4, pQE-lyz1), the strain was cultured at 37 °C in 400 ml Luria Bertani (LB) medium with Ap (100 µg ml–1) and Km (20 µg ml–1), to an OD600 of 0·5. The culture was induced with 1 mM IPTG and further incubated for 1 h without shaking, then centrifuged at 4000 g for 20 min, and the pellet was suspended in 2·5 ml buffer A [10 nM imidazole, 0.3 M NaCl, 50 nM NaH2PO4 (pH 8.0)], which is recommended for Ni columns (Qiagen). After sonication, a clear cell lysate was obtained by centrifugation at 10 000 g for 20 min. The lysozyme activity was screened as a growth inhibition zone on a lawn of Ea1/79 after application of 10 µl cell lysate. The protein concentration was measured by the Lowry method.

Lysozyme assays by inhibition of cell growth in culture.
Lysozyme activity was assayed with Erw. amylovora Ea1/79Sm grown in LB medium overnight at 28 °C. The cells were diluted to 105, 104, 103 and 102 c.f.u. ml–1 and dispensed in triplicate into 96-well microtitre plates (200 µl per well). The sonicated lysates of M15(pREP4, pQE-lyz1) were added at various concentrations to Ea1/79Sm dilutions, which were further incubated at 28 °C in LB medium with Sm for up to 3 days without shaking. For a negative control, protein from IPTG-induced M15(pREP4, pQE-30) lysate was applied. The bacterial growth rate was estimated by automatic OD620 measurements using a Titertek Multiskan MCC/340 MKII (Flow Laboratories).

For survival assays, suspensions in Standard I broth (StI, Merck) were incubated in a microtitre plate for 0, 1, 2, or 4 h, and the reaction mixtures were plated on StI agar with 200 µg Sm ml–1.

Assays with pear slices.
Immature pears (cv. Bartlett) were stored for 1–10 weeks in loosely sealed glass beakers at 6 °C. Slices of approximately 5 mm thickness were cut and briefly immersed in cleared cell lysates, which had been obtained by sonication and subsequent centrifugation. The extracts were diluted in imidazole-containing buffer to a protein concentration of 500 µg ml–1. The soaked slices were air-dried in a laminar flow hood, and four slices were placed into a plastic box (5 cm diameter). With a pipette, 10 µl volumes of dilutions of Erw. amylovora cultures were applied to the surface of the four slices in the box, which was then tightly closed and incubated at 26 °C. Necrosis and ooze formation were evaluated after 5 days with a scale from 0 (no symptoms) to 4 (dark brown slices and ooze in large drops or as a fluid layer on the surface).

Analysis of {phi}Ea1h lysozyme on Ni columns.
Soluble extracts from M15(pREP4, pQE-lyz1) were loaded on an Ni-NTA agarose column (Qiagen). The column was washed with washing buffer (1x PBS with 0·5 M NaCl, 0·05 % Tween 20, 5 mM imidazole, pH 8) and eluted with the same buffer containing 250 mM imidazole (pH 6·5). The columns were then treated with 0·1 M EDTA, and the matrix was further analysed for residual protein.

PAGE analysis and immunoblots.
Fractions from each step of the purification procedure were separated by size in 14 % polyacrylamide gels using a discontinuous buffer system (Laemmli, 1970) and transferred to PVDF membranes (Millipore) in a Hoefer Mighty Small SE245 system as recommended by the manufacturer. The blots were treated by standard techniques, first using an anti-His antibody (Qiagen), and then an anti-mouse antibody conjugated with alkaline phosphatase. The colour detection of the immobilized antigen was done with NBT/BCIP (Pierce, Perbio Science).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sequence analysis of ORF178 and ORF119
The fragment in plasmid pJH94 derived from a partial Sau3A digest was previously sequenced (GenBank accession no. AJ278614) and three ORFs were identified: one encoded an exopolysaccharide depolymerase (Kim & Geider, 2000) and the two other ORFs were tentatively assigned to encode a lysozyme (ORF178) and a holin (ORF119). ORF119 overlapped by 19 bp with the 3' end of ORF178, indicating that its expression might rely on the promoter of ORF178, which is apparently transcribed by an RNA polymerase encoded or modified by the bacteriophage. The potential ribosome-binding sites (RBSs) AGGAGG and AGGACA were found upstream of ORF178 and ORF119, respectively (Fig. 1), which are in agreement with the prokaryotic consensus sequence. The palindromic sequence ACAGGNACC-n6-GGTNCCTGT, which may serve as a terminator, was identified behind ORF119.



View larger version (58K):
[in this window]
[in a new window]
 
Fig. 1. Nucleotide sequence of a DNA fragment with ORF178 and ORF119 indicating control elements for the lyz and hol genes, respectively. RBS, ribosome-binding site.

 
Further sequence database searches with BLASTP+BEAUTY revealed that the protein encoded by ORF178 had significant homology with the lysozymes of Haemophilus influenzae phage HP1 (53 % similarity, 36 % identity; Benjamin et al., 1984; Esposito et al., 1996), E. coli phage P1 (51 % similarity, 33 % identity; Schmidt et al., 1996), APSE-1, a bacteriophage from an endosymbiont of the aphid Acyrthosiphon pisum (49 % similarity, 33 % identity; van der Wilk et al., 1999), and Salmonella typhimurium phage P22 (55 % similarity, 37 % identity; Rennell & Poteete, 1985). The lysozymes from HP1, P1 (protein gp17), P22 (gp19) and APSE-1 (endolysin P13) share structurally conserved regions marked in the alignment with ORF178 (Fig. 2).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2. Alignment of the amino acid sequence of ORF178 ({phi}Ea1_lyz) with viral lysozymes. The small boxes indicate regions with high similarity (>70%). {phi}Ea1_lyz, EBI/UniProt accession no. Q9FZS7; P1_gp17, accession no. Q37875; HP1_lys, accession no. P51728; P22_gp19, accession no. P09963; APSE_P13, accession no. Q9T1T5.

 
Cloning and expression of the viral lysozyme gene in E. coli
The lyz gene was amplified by PCR without the start codon and cloned into plasmid pQE-30 as an N-terminal His-tag fusion. The resulting plasmid pQE-lyz1 was introduced into E. coli M15(pREP4) and lysozyme expression was induced by addition of IPTG (Fig. 3). The cell growth of the IPTG-treated E. coli strain M15(pREP4, pQE-lyz1) was inhibited and the culture showed symptoms of cell lysis, which was in contrast to M15(pREP4, pQE-30), where no differences were found for the growth of non-induced and induced cultures. Supernatants of induced M15(pREP4, pQE-lyz1) cells and the cell pellet, resuspended in buffer A, were applied to the Ni column and analysed by Western blotting for lysozyme expression. No lysozyme was detected in the flowthrough, the wash or the elution fractions, including treatment with 0·1 M EDTA. However, a signal was detected by Western blot analysis after boiling the matrix in sample buffer. When aliquots of the column matrix were applied to Ea1/79 cells forming a lawn on agar, a growth inhibition zone was obtained due to the release of lysozyme (similar to the pattern in Fig. 4).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3. Growth inhibition by induction of lysozyme expression in E. coli. Cultures of M15(pREP4) with pQE-30 (continuous line, solid symbols) or pQE-lyz1 (dashed line, open symbols) were grown at 37 °C to mid-exponential phase (OD620, 0·5–0·7). Untreated cultures ({bullet}, {circ}) or cultures induced by addition of IPTG to a final concentration of 1 mM ({blacktriangleup}, {triangleup}) were monitored for growth. The experiments were repeated at least three times with similar results.

 


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 4. Lysozyme activity on a lawn of Ea1/79Sm. Cell lysates (10 µl with 120 µg protein) in (A) are from the induced strain M15(pREP4, pQE-lyz1) after growth in LB with Ap and Km and those in (B) are from the induced strain M15(pREP4-Cm, pQE-lyz1) after growth in LB with Ap and Cm.

 
Requirements for cloning and enzyme expression
Host requirements for expression of the lysozyme gene were assayed by transfer of plasmids prepared from M15(pREP4, pQE-lyz1) into various E. coli strains. When selection was done with Ap only, all cells recovered contained the plasmids pREP4 and pQE-lyz1, indicating the need for a high level of lac repressor for cells with pQE-lyz1. Among several E. coli strains tested, only the commercial strains M15 and GI698 were successfully transformed with pQE-lyz1 and pREP4 for expression of lysozyme, which depended strictly on Km in the growth medium (Table 2). When M15(pREP4, pQE-lyz1) was grown in Ap without Km, the lysozyme was barely recovered in induced cell lysates, although plasmid pREP4 was not lost. When pREP4 was altered by replacing the Km-resistance gene with a Cm cassette, lysozyme expression was not detected after induction of a culture grown in Ap and Cm (Fig. 4B). Activity of M15(pREP4-Cm, pQE-lyz1) lysates was restored when the cells were transformed with another compatible plasmid carrying Km resistance, such as pfdC1 or pRK293 (Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2. Bacterial strains and plasmids for expression of the lysozyme gene

 
Determination of lysozyme activity by cell growth
After induction with IPTG, cleared cell lysates were prepared from cultures of M15(pREP4, pQE-lyz1), and then tested for formation of inhibition zones on a lawn of Ea1/79 cells (Fig. 4A). The lysates showed haloes on the agar. A quantitative assay was developed by growth-inhibition experiments with cell cultures in the presence of lysozyme. Titration with a constant amount of protein of the cell lysate showed growth inhibition, which was dependent on the initial concentration of Ea1/79Sm. For 105 c.f.u. ml–1, the optical density was approximately half that of the control after 48 h. An even greater growth inhibition was observed for 104 c.f.u. ml–1 starting culture, and growth was completely inhibited when 103 c.f.u. ml–1 or less was applied (Fig. 5). Control cell lysates did not affect growth of Erw. amylovora.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. Influence of lysozyme on the growth of Erw. amylovora. Aliquots with 25 µg protein of cleared E. coli cell lysates were added to 200 µl suspensions of Erw. amylovora strain Ea1/79Sm. Continuous line, solid symbols: supernatant from IPTG-induced E. coli M15(pREP4, pQE-30) was the control. Dashed line, open symbols: cell lysates of IPTG-induced M15(pREP4,pQE-lyz1). {bullet}, {circ}, 105 c.f.u. ml–1; {blacktriangleup}, {triangleup}, 104 c.f.u. ml–1; {blacksquare}, {square}, 103 c.f.u. ml–1; {blacklozenge}, {lozenge}, 102 c.f.u. ml–1 of Erw. amylovora strain Ea1/79Sm. The experiments were repeated at least three times with similar results.

 
Increasing amounts of the cell lysate from induced E. coli M15(pREP4, pQE-lyz1) were added to 200 µl Ea1/79Sm culture, diluted to 105 c.f.u. ml–1, and the effect on cell growth was measured by turbidity. Growth inhibition was visible for protein concentrations in the range of 5–30 µg, whereas the addition of control lysates had no effect on cell growth (Fig. 6). Addition of 30 µg protein from a M15(pREP4, pQE-lyz1) lysate completely abolished cell growth, and plating of culture aliquots after incubation for 72 h did not result in colony formation on agar (data not shown).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6. Growth inhibition of Erw. amylovora cells at different lysozyme concentrations. A cell lysate from IPTG-induced M15(pREP4,pQE-lyz1) was diluted to protein concentrations of 5 ({circ}), 11 ({triangleup}), 16 ({square}), 22 ({lozenge}) and 27 µg (x) and added to 200 µl suspensions of Erw. amylovora strain Ea1/79Sm diluted in LB medium to 105 c.f.u. ml–1. Cell lysate with 30 µg protein from IPTG-induced E. coli M15(pREP4, pQE-30) ({bullet}) was used as the control. The experiments were repeated at least three times with similar results.

 
Incubation of Ea1/79Sm cells with extracts containing lysozyme resulted in a decrease of their viability (Table 3). After applying 36 µg protein for 4 h, a strong decrease in viability was achieved. A similar effect was observed for 72 µg protein and either 2 or 4 h incubation. We assume that the amount of lysozyme must exceed a threshold in order to inhibit the applied cells.


View this table:
[in this window]
[in a new window]
 
Table 3. Effect of lysozyme on viability of Erw. amylovora

Various amounts of extract of induced M15(pREP4, pQE-lyz1) cells were added to 200 µl overnight cultures of 200 c.f.u. Ea1/79Sm. The control was an extract from M15(pREP4, pQE-30).

 
Virulence assays on pear slices treated with lysozyme-containing cell lysates
Slices from immature pears were soaked in cleared extracts of induced and sonicated cells from M15(pREP4, pQE-lyz1) or M15(pREP4, pQE-30). They were briefly dried, then inoculated with 10 µl diluted overnight cultures of Ea1/79 by applying tenfold dilutions from 5x104 to 5x101 c.f.u. per slice. For lysozyme-containing cell lysates, weak symptoms were found at the highest inoculation density, but none were found when lower amounts of Erw. amylovora cells were applied (results not shown). With cell lysates of M15(pREP4, pQE-30) as control, typical fire blight symptoms, such as ooze formation and necrosis, were observed. As for the cell-damaging effects of lysozyme in bacterial suspensions, the enzyme showed similar effects when applied to pear slices.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The expression of various lytic proteins has been attempted in transgenic plants for control of fire blight. The bacteriophage T4 lysozyme gene was introduced into potato to control soft rot caused by Erwinia carotovora (Düring et al., 1993), and also into apple (Hanke et al., 1998); a marginal reduction of symptom formation was described, but this might be surpassed by haemolytic proteins such as attacin (Reynoird et al., 1999; Ko et al., 2002) and cecropin (Mourgues et al., 1998). The haemolytic protein genes are some of the defence mechanisms used by insects to attack microbial pathogens; their mode of action is disruption of membrane integrity (Engström et al., 1984; Boman & Hultmark, 1987). The T4 lysozyme was combined with attacin to enhance fire blight resistance of transgenic apples but a synergistic effect was not observed (Ko et al., 1999, 2002).

It has recently been suggested that attacin and cecropin might also affect mammals and might be harmful in transgenic food. Hen egg white contains a significant amount of lysozyme, which is a component of daily human nutrition. Viral lysozymes are produced in bacteria as tools for destruction of the host cell walls; they lack signal peptides and cannot be secreted into the periplasm. The pore-forming holins support lysozyme transport after simultaneous gene expression in the life cycle of bacteriophages. When the lysozyme is expressed in transgenic bacteria or supplied to cultured cells, the protein has been suggested to act via a membrane-disrupting activity (Düring et al., 1999). Although the muramidase activity of lysozyme damage to the peptidoglycan layer is well documented, the protein, variants or even fragments, may also interact non-enzymically with the bacterial membrane (Düring et al., 1999; Pellegrini et al., 2000). This additional bactericidal activity could be an important feature of the {phi}Ea1h lysozyme (H. Salm & K. Geider, unpublished). Bactericidal activities of proteins are quite common, and have also been suggested for lactoferrin (Zhang et al., 1998); however, this effect might be different from the muramidase activity of lysozyme. In this study, we have cloned and partially characterized a lysozyme from Erw. amylovora phage {phi}Ea1h. Many elaborate lysozyme assays have been published (Saedi et al., 1987; van de Guchte et al., 1992; Pontarollo et al., 1997; Wang & Chag, 1997). However, the outer membrane of Gram-negative bacteria prevents lysozyme from easily accessing the peptidoglycan layer. Therefore, in the above lysozyme assays, indicator cells were treated with EDTA or chloroform to facilitate invasion of lysozyme to the peptidoglycan layer, and the decrease in turbidity of the cell suspension was then determined. This application was not appropriate for Erw. amylovora because the EDTA-treated cells quickly lyse. Growth assays of broth cultures in microtitre plates and growth inhibition on a bacterial lawn were convenient tools to determine lysozyme activity in the present study.

Although the antibacterial activity was measured in lysates of induced M15(pREP4, pQE-lyz1) cells with several different assays, no signals were detected in Western blots with these protein fractions. Nevertheless, the matrix of the Ni column was positive in the immunological assay, and showed bactericidal activity on plates. Consequently, the amount of lysozyme in cell lysates was too low for protein detection in a Western blot, but sufficient for formation of inhibition zones on a bacterial cell lawn.

After induction of lysozyme expression, host cell growth was abolished and the bacteria were lysed. Since lysozyme seems to be toxic to host cells even at low levels, promoters for lyz expression must be tightly downregulated during cell growth. This was attempted with the lac repressor constitutively expressed from a plasmid. Even in this case, expression in E. coli was not always possible, and only two strains synthesized detectable amounts of lysozyme. This could be due to selection of the commercial strains for tolerance to foreign proteins expressed from cloned genes. Another requirement for lyz expression was host cell resistance to Km, and growth of the cells in the presence of the antibiotic. Km could act as an inhibitor of the enzyme by binding to {phi}Ea1h lysozyme. A similar effect was detected for hen egg white lysozyme activity and several aminoglycosidic antibiotics including Km A (Fernández-Sousa et al., 1977). The inhibition effect was explained by the related structure of aminoglycosidic antibiotics and the saccharidic lysozyme substrate.

Large-scale production of cell extracts enriched for {phi}Ea1h lysozyme would allow attempts to protect orchards against fire blight by spraying. The gene could also be expressed in fire-blight host plants to release the protein after plant-cell damage by the pathogen. Adjacent to the lyz gene on the genome of bacteriophage {phi}Ea1h, an ORF encoding exopolysaccharide depolymerase was cloned (Vandenbergh et al., 1985; Vandenbergh & Cole, 1986; Hartung et al., 1988) and further characterized (Kim & Geider, 2000). Expression of the exopolysaccharide depolymerase in plants was attempted in order to degrade the amylovoran capsules of Erw. amylovora, thus exposing the pathogen to plant defence reactions. First reports suggest a positive role of dpo expression in apples and pears, with reduced fire blight symptoms of selected cell lines (Hanke et al., 2003; M. Malnoy, M. Faize, J. S. Venisse, K. Geider & E. Chevreau, unpublished). Coexpression of the depolymerase and the lysozyme of Erw. amylovora phage {phi}Ea1h in plant cells could result in synergistic effects of the two proteins in the control of colonization by the fire blight pathogen in plant tissue.


   ACKNOWLEDGEMENTS
 
We thank B. Schneider, BBA Dossenheim, for experimental support in Western blots and for comments on the manuscript.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Benjamin, R. C., Fitzmaurice, W. P., Huang, P. C. & Scocca, J. J. (1984). Nucleotide sequence of cloned DNA segments of the Haemophilus influenzae bacteriophage HP1c1. Gene 31, 173–185.[CrossRef][Medline]

Bernhard, F., Coplin, D. L. & Geider, K. (1993). A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii. Mol Gen Genet 239, 158–168.[Medline]

Billing, E. (1960). An association between capsulation and phage sensitivity in Erwinia amylovora. Nature 186, 819–820.[Medline]

Boman, H. G. & Hultmark, D. (1987). Cell-free immunity in insects. Annu Rev Microbiol 41, 103–126.[CrossRef][Medline]

Bonn, W. G. & van der Zwet, T. (2000). Distribution and economic importance of fire blight. In Fire Blight: the Disease and its Causative Agent Erwinia amylovora, pp. 37–53. Edited by J. L. Vanneste. Wallingford, UK & New York: CABI Publishing.

Cooper, G. M. (1997). The Cell: a Molecular Approach. Washington, DC: American Society for Microbiology.

Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X. W., Finlay, D. R., Guiney, D. & Helsinki, D. R. (1985). Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene-expression. Plasmid 13, 149–153.[Medline]

Düring, K., Porsch, P., Fladung, M. & Lörz, H. (1993). Transgenic potato plants resistant to the phytopathogenic bacterium Erwinia carotovora. Plant J 3, 587–598.[CrossRef]

Düring, K., Porsch, P., Mahn, A., Brinkmann, O. & Gieffers, W. (1999). The non-enzymatic microbicidal activity of lysozymes. FEBS Lett 449, 93–100.[CrossRef][Medline]

Engström, P., Carlsson, A., Engström, A., Zao, Z. J. & Bennich, H. (1984). The antibacterial effect of attacins from the silk moth Hyalophora cecropia is directed against the outer membrane of Escherichia coli. EMBO J 3, 3347–3351.[Abstract]

Erskine, J. M. (1973). Characteristics of Erwinia amylovora bacteriophage and its possible role in the epidemology of fire blight. Can J Microbiol 19, 837–845.[Medline]

Esposito, D., Fitzmaurice, W. P., Benjamin, R. C., Goodman, S. D., Waldman, A. S. & Scocca, J. J. (1996). The complete nucleotide sequence of bacteriophage HP1 DNA. Nucleic Acids Res 24, 2360–2368.[Abstract/Free Full Text]

Falkenstein, H., Bellemann, P., Walter, S., Zeller, W. & Geider, K. (1988). Identification of Erwinia amylovora, the fire blight pathogen, by colony hybridization with DNA from plasmid pEA29. Appl Environ Microbiol 54, 2798–2802.

Fernández-Sousa, J. M., Gavilanes, J. G., Municio, A. M. & Rodriguez, R. (1977). On the inhibition of hen egg-white lysozyme activity by aminoglycosidic antibiotics. Biochem Biophys Res Commun 75, 895–900.[Medline]

Geider, K., Baldes, R., Bellemann, P., Metzger, M. & Schwartz, T. (1995). Mutual adaptation of bacteriophage fd, pfd plasmids and their host strains. Microbiol Res 150, 337–346.[Medline]

Hanke, V., Norelli, J. L., Aldwinckle, H. S. & Düring, K. (1998). Transformation of apple cultivars with T4-lysozyme gene to increase fire blight resistance. Acta Hortic 489, 253–256.

Hanke, V., Geider, K. & Richter, K. (2003). Transgenic apple plants expressing viral EPS-depolymerase: evaluation of resistance to the phytopathogenic bacterium Erwinia amylovora. In Plant Biotech 2002 and Beyond, pp. 153–157. Edited by I. K. Vasil. Dordrecht: Kluwer.

Hartung, J. S., Fulbright, D. W. & Klos, E. J. (1988). Cloning of a bacteriophage polysaccharide depolymerase gene and its expression in Erwinia amylovora. Mol Plant–Microbe Interact 1, 87–93.

Hildebrand, M., Dickler, E. & Geider, K. (2000). Occurrence of Erwinia amylovora on insects in a fire blight orchard. J Phytopathol 148, 251–256.[CrossRef]

Jabrane, A., Sabri, A., Compère, P., Jacques, P., Vandenberghe, I., Van Beeumen, J. & Thonart, P. (2002). Characterization of Serracin P, a phage-tail-like bacteriocin, and its activity against Erwinia amylovora, the fire blight pathogen. Appl Environ Microbiol 68, 5704–5710.[Abstract/Free Full Text]

Jock, S. & Geider, K. (2004). Molecular differentiation of Erwinia amylovora strains from North America and of two Asian pear pathogens by analyses of PFGE patterns and hrpN genes. Environm Microbiol 6, 480–490.[CrossRef]

Jolles, P. & Jolles, J. (1984). What's new in lysozyme research? Always a model system, today as yesterday. Mol Cell Biochem 63, 165–189.[Medline]

Kim, W.-S. & Geider, K. (2000). Characterization of a viral EPS-depolymerase, a potential tool for control of fire blight. Phytopathology 90, 1263–1268.

Ko, K., Norelli, J. L., Brown, S. K., Borejsza-Wysocka, S. K., Düring, K. & Aldwinckle, H. S. (1999). Effects of multiple transgenes on resistance to fire blight of ‘Galaxy’ apple. Acta Horticult 489, 257–257.

Ko, K., Norelli, J. L., Reynord, J.-P., Aldwinckle, H. S. & Brown, S. K. (2002). T4 lysozyme and attacin genes enhance resistance of transgenic ‘Galaxy’ apple against Erwinia amylovora. J Am Soc Hortic Sci 127, 515–519.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[Medline]

Mourgues, F., Brisset, M. N. & Chevreau, E. (1998). Strategies to improve plant resistance to bacterial diseases through genetic engineering. Trends Biotechnol 16, 203–210.[CrossRef][Medline]

Okabe, N. & Goto, M. (1963). Bacteriophage of plant pathogens. Annu Rev Phytopathol 1, 397–418.[CrossRef]

Pellegrini, A., Thomas, U., Wild, P., Schraner, E. & von Fellenberg, R. (2000). Effect of lysozyme or modified fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli. Microbiol Res 155, 69–77.[Medline]

Pontarollo, R. A., Rioux, C. R. & Potter, A. A. (1997). Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1. J Bacteriol 179, 1872–1879.[Abstract]

Psallidas, P. G. & Tsiantos, J. (2000). Chemical control of fire blight. In Fire Blight, the Disease and its Causative Agent, Erwinia amylovora, pp. 199–234. Edited by J. L. Vanneste. Wallingford, UK & New York: CABI Publishing.

Rennell, D. & Poteete, A. R. (1985). Phage P22 lysis genes: nucleotide sequences and functional relationships with T4 and lambda genes. Virology 143, 280–289.[CrossRef][Medline]

Reynoird, J. P., Mourgues, F., Norelli, J., Aldwinckle, H. S., Brisset, M. N. & Chevreau, E. (1999). First evidence for improved resistance to fire blight in transgenic pear expressing the attacin E gene from Hyalophora cecropia. Plant Science 149, 23–31.[CrossRef]

Ritchie, D. F. & Klos, E. J. (1977). Isolation of Erwinia amylovora bacteriophage from aerial parts of apple trees. Phytopathology 67, 101–104.

Saedi, M. S., Garvey, K. J. & Ito, J. (1987). Cloning and purification of a unique lysozyme produced by Bacillus phage {phi}29. Proc Natl Acad Sci U S A 84, 955–958.[Abstract]

Schmidt, C., Velleman, M. & Arber, W. (1996). Three functions of bacteriophage P1 involved in cell lysis. J Bacteriol 178, 1099–1104.[Abstract]

Schnabel, E. L. & Jones, A. L. (2001). Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora. Appl Environ Microbiol 67, 59–64.[Abstract/Free Full Text]

Van de Guchte, M., van der Wal, F. J. & Venema, G. (1992). Lysozyme expression in Lactococcus lactis. Appl Microbiol Biotechnol 37, 216–224.[Medline]

Vandenbergh, P. A. & Cole, R. L. (1986). Cloning and expression in Escherichia coli of the polysaccharide depolymerase associated with bacteriophage-infected Erwinia amylovora. Appl Environ Microbiol 51, 862–864.

Vandenbergh, P. A., Wright, A. M. & Vidaver, A. K. (1985). Partial purification and characterization of a polysaccharide depolymerase associated with phage-infected Erwinia amylovora. Appl Environ Microbiol 49, 994–996.

Van der Wilk, F., Dullemans, A. M., Verbeek, M. & van den Heuvel, J. F. (1999). Isolation and characterization of APSE-1, a bacteriophage infecting the secondary endosymbiont of Acyrthosiphon pisum. Virology 262, 104–113.[CrossRef][Medline]

Wang, S.-L. & Chag, W.-T. (1997). Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl Environ Microbiol 63, 380–386.[Abstract]

Weaver, L. H., Rennell, D., Poteete, A. R. & Mathews, B. W. (1985). Structure of phage P22 gene 19 lysozyme inferred from its homology with phage T4 lysozyme. Implications for lysozyme evolution. J Mol Biol 184, 739–741.[Medline]

Young, R. (1992). Bacteriophage lysis: mechanism and regulation. Microbiol Rev 56, 430–481.[Medline]

Zhang, Z. Y., Coyne, D. P., Vidaver, A. K. & Mitra, A. (1998). Expression of human lactoferrin cDNA confers resistance to Ralstonia solanacearum in transgenic tobacco plants. Phytopathology 88, 730–734.

Received 8 April 2004; revised 6 May 2004; accepted 6 May 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Kim, W.-S.
Articles by Geider, K.
Articles citing this Article
PubMed
PubMed Citation
Articles by Kim, W.-S.
Articles by Geider, K.
Agricola
Articles by Kim, W.-S.
Articles by Geider, K.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.