Articles
INTERACTION OF VIRAL AND BACTERIAL LYSOZYMES WITH ERWINIA AMYLOVORA AND THEIR INHIBITION BY A BACTERIAL PROTEIN
Article number
896_60
Pages
421 – 423
Language
English
Abstract
Viral lysozymes can interact with Erwinia amylovora, and their genes may control fire blight when expressed in the pathogen.
We have investigated lysozymes from E. amylovora phages and from genomes of several Erwinia species.
With chloroform-treated cells as substrate, the highest lytic activity was found for viral lysozymes.
Fusion proteins with the ΦEa1h lysozyme, such as addition of a His-tag reduced lysis efficiency significantly.
For large fusions at the N- or C-terminus lytic activity was completely lost.
When a protein fused to glutathione S-transferase was cleaved off with a specific protease, the activity of the viral lysozyme was restored.
Intact lysozyme from E. amylovora phage ΦEa1h had a similar lytic activity as E. coli phage T4 lysozyme.
Viable cells of E. amylovora were not inhibited by external addition of lysozyme from sonicated cell extracts in assays with ΦEa1h lysozyme and T4 lysozyme.
The cellular expression of a viral lysozyme gene can be toxic for a bacterial host.
The gene was therefore expressed in yeast as safe environment for its secretion.
Lysozyme-specific antibodies detected the protein in culture supernatants of transgenic yeast.
When E. amylovora was transformed with a viral lysozyme gene, the cells were killed after induction by IPTG. Transfer of a lyz-gene into E. amylovora can thus strongly reduce the viability of the pathogen.
The ΦEa1h lysozyme was inhibited by a cellular protein from E. amylovora related to an inhibitor for vertebrate lysozymes of E. coli. The lytic activity of lysozyme from phage ΦEa104 and ΦEa116 was not affected by the enzyme inhibitor.
We have investigated lysozymes from E. amylovora phages and from genomes of several Erwinia species.
With chloroform-treated cells as substrate, the highest lytic activity was found for viral lysozymes.
Fusion proteins with the ΦEa1h lysozyme, such as addition of a His-tag reduced lysis efficiency significantly.
For large fusions at the N- or C-terminus lytic activity was completely lost.
When a protein fused to glutathione S-transferase was cleaved off with a specific protease, the activity of the viral lysozyme was restored.
Intact lysozyme from E. amylovora phage ΦEa1h had a similar lytic activity as E. coli phage T4 lysozyme.
Viable cells of E. amylovora were not inhibited by external addition of lysozyme from sonicated cell extracts in assays with ΦEa1h lysozyme and T4 lysozyme.
The cellular expression of a viral lysozyme gene can be toxic for a bacterial host.
The gene was therefore expressed in yeast as safe environment for its secretion.
Lysozyme-specific antibodies detected the protein in culture supernatants of transgenic yeast.
When E. amylovora was transformed with a viral lysozyme gene, the cells were killed after induction by IPTG. Transfer of a lyz-gene into E. amylovora can thus strongly reduce the viability of the pathogen.
The ΦEa1h lysozyme was inhibited by a cellular protein from E. amylovora related to an inhibitor for vertebrate lysozymes of E. coli. The lytic activity of lysozyme from phage ΦEa104 and ΦEa116 was not affected by the enzyme inhibitor.
Publication
Authors
K. Geider, B. Schneider, I. Müller
Keywords
cell lysis, plasmid transfer, fusion proteins, Ivy
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