Max-Planck-Institut für Zellbiologie, Ladenburg, Germany
Correspondence
Klaus Geider
k.geider{at}bba.de
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ABSTRACT |
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INTRODUCTION |
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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-
-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 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.
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METHODS |
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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 ml1) and Km (20 µg ml1), 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. ml1 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 ml1.
Assays with pear slices.
Immature pears (cv. Bartlett) were stored for 110 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 ml1. 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 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).
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RESULTS |
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DISCUSSION |
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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
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
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 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 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
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
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.
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ACKNOWLEDGEMENTS |
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Received 8 April 2004;
revised 6 May 2004;
accepted 6 May 2004.
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