Molecular characterization of a protease secreted by Erwinia amylovora.
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.
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Max-Planck-Institut für Zellbiologie, Rosenhof, Ladenburg, D-68526, Germany.
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D000595:Amino Acid Sequence; D003470:Culture Media; D004251:DNA Transposable Elements; D004885:Erwinia / Q000201:enzymology* / Q000472:pathogenicity / Q000502:physiology; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D018515:Plant Leaves; D019685:Rosales / Q000382:microbiology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014774:Virulence; D015032:Zinc
D003470:Culture Media; D004251:DNA Transposable Elements; D008666:Metalloendopeptidases; C079508:PrtA metalloprotease; D015032:Zinc