Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene.
The prt1 gene encoding extracellular protease from Erwinia carotovora subsp. carotovora EC14 in cosmid pCA7 was subcloned to create plasmid pSK1. The partial nucleotide sequence of the insert in pSK1 (1,878 bp) revealed a 1,041-bp open reading frame (ORF1) that correlated with protease activity in deletion mutants. ORF1 encodes a polypeptide of 347 amino acids with a calculated molecular mass of 38,826 Da. Escherichia coli transformed with pSK1 or pSK23, a subclone of pSK1, produces a protease (Prt1) intracellularly with a molecular mass of 38 kDa and a pI of 4.8. Prt1 activity was inhibited by phenanthroline, suggesting that it is a metalloprotease. The prt1 promoter was localized between 173 and 1,173 bp upstream of ORF1 by constructing transcriptional lacZ fusions. Primer extension identified the prt1 transcription start site 205 bp upstream of ORF1. The deduced amino acid sequence of ORF1 showed significant sequence identity to metalloproteases from Bacillus thermoproteolyticus (thermolysin), B. subtilis (neutral protease), Legionella pneumophila (metalloprotease), and Pseudomonas aeruginosa (elastase). It has less sequence similarity to metalloproteases from Serratia marcescens and Erwinia chrysanthemi. Locations for three zinc ligands and the active site for E. carotovora subsp. carotovora protease were predicted from thermolysin.
Kyöstiö
S R
SR
Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Cramer
C L
CL
Lacy
G H
GH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D007525:Isoelectric Focusing; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D016973:Pectobacterium carotovorum / Q000201:enzymology* / Q000235:genetics; D010618:Phenanthrolines / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011480:Protease Inhibitors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D016415:Sequence Alignment; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D009841:Oligonucleotides; D010618:Phenanthrolines; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D010450:Endopeptidases; D008666:Metalloendopeptidases; C071300:Prt1 protease