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Molecular characterization of a protease secreted by Erwinia amylovora
1Max-Planck-Institut für Zellbiologie, Rosenhof, Ladenburg, D-68526, Germany.
Abstract:
A protease with a molecular mass of 48 kDa is secreted by the fire blight pathogen Erwinia amylovora in minimal medium. We characterized this activity as a metalloprotease, since the enzyme was inhibited by EDTA and o -phenanthroline. A gene cluster was determined to encode four genes connected to protease expression, including a structural gene (prtA) and three genes (prtD, prtE, prtF) for secretion of the protease, which are transcribed in the same direction. The organization of the protease gene cluster in E. amylovora is different from that in other Gram-negative bacteria, such as Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens. On the basis of the conservative motif of metalloproteases, PrtA was identified to be a member of the metzincin subfamily of zinc-binding metalloproteases, and was confirmed to be the 48 kDa protease on gels by sequencing of tryptic peptide fragments derived from the protein. The protease is apparently secreted into the external medium through the type I secretion pathway via PrtD, PrtE and PrtF which share more than 90% identity with the secretion apparatus for lipase of S. marcescens. A protease mutant was created by Tn 5 -insertions, and the mutation localized in the prtD gene. The lack of protease reduced colonization of an E. amylovora secretion mutant labelled with the gene for the green fluorescent protein (gfp) in the parenchyma of apple leaves.
Insights
Erwinia amylovora secretes a 48 kDa metalloprotease involved in plant pathogen colonization. This protease, encoded by the prtA gene and secreted via a type I pathway, is crucial for fire blight disease development in apple leaves.
Area of Science:
- Microbiology
- Plant Pathology
- Biochemistry
Background:
- Erwinia amylovora is the causal agent of fire blight, a destructive disease affecting apple and pear trees.
- Proteases secreted by plant pathogens often play significant roles in virulence and disease development.
Purpose of the Study:
- To characterize the secreted protease from Erwinia amylovora.
- To identify the genes involved in protease expression and secretion.
- To investigate the role of the protease in E. amylovora colonization of apple leaves.
Main Methods:
- Biochemical characterization of protease activity (inhibition by EDTA, o-phenanthroline).
- Genetic analysis of the protease gene cluster (prtA, prtD, prtE, prtF).
- Identification of PrtA as a metzincin metalloprotease via peptide sequencing.
- Construction of a protease mutant using Tn5 insertion.
- Assessment of mutant colonization in apple leaf parenchyma using GFP labeling.
Main Results:
- A 48 kDa metalloprotease was identified and characterized in E. amylovora.
- A four-gene cluster (prtA, prtD, prtE, prtF) was identified, encoding the protease and its type I secretion system.
- PrtA belongs to the metzincin subfamily of metalloproteases.
- A prtD mutant exhibited reduced colonization of apple leaf parenchyma.
Conclusions:
- The identified metalloprotease and its type I secretion system are important for Erwinia amylovora virulence.
- The protease contributes to the colonization of apple leaves, suggesting a role in fire blight pathogenesis.