Erwinia carotovora subsp. carotovora extracellular protease: characterization and nucleotide sequence of the gene

Abstract

The prtl 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 pSKI or pSK23, a subclone of pSK1, produces a protease (Prtl) intracellularly with a molecular mass of 38 kDa and a pI of 4.8. Prtl activity was inhibited by phenanthroline, suggesting that it is a metalloprotease. The prtl promoter was localized between 173 and 1,173 bp upstream of ORF1 by constructing transcriptional lacZ fusions. Primer extension identified the prtl 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.Erwinia carotovora subsp. carotovora EC14 is a gramnegative bacterium which causes soft rot on many plant species (39). Soft rot of potato (Solanum tuberosum) tubers is associated with the production of several degradative enzymes secreted by E. carotovora subsp. carotovora, including pectolytic enzymes, cellulases, proteases, and phospholipases (10, 57). Pectolytic enzymes probably play the most important role in maceration by degrading pectic components of the plant cell wall and middle lamella, resulting in separation of the cells (5). Possible roles for other degradative enzymes have not been established.Several erwinias and pseudomonads causing soft rot secrete proteases. Among soft-rot pseudomonads, extracellular protease production correlates more strongly with the ability to macerate plant tissue than does pectolytic enzyme production (46). In E. carotovora subsp. carotovora, large amounts of extracellular protease are produced when the bacterium is grown in rich broth, on bean (Phaseolus vulgaris) hypocotyls, or on sliced cucumber (Cucumis sativus) fruit (57), but the physiological role of the protease is unknown. Protease may aid in the degradation of plant cell wall components, cytoplasmic membranes, or cytosolic proteins. Purified E. carotovora subsp. carotovora protease causes limited cell death on cucumber disks and lysis of cucumber protoplasts (57). The release of amino acids and small peptides by E. carotovora subsp. carotovora protease may increase the rate of bacterial growth, thereby increasing the ability of the pathogen to colonize its host. The nutritional benefit derived from the proteolytic digestion of host macromolecules may contribute to greater virulen...

Characterization of the Zinc-Containing Metalloprotease Encoded by zpx and Development of a Species-Specific Detection Method for Enterobacter sakazakii

“…38,000 ( Fig. 2A), which is similar to the molecular sizes of proteases that are described for other bacterial pathogens (5,21). However, it is 10 kDa smaller than the proteases produced by Erwinia amylovora (35) and V. vulnificus (24).…”

“…2B, panel a) revealed the presence of a single band with 4.4 as an isoelectric point of the native protein. This value is lower than pIs reported for various other proteases, such as those expressed by several marine vibrio species (5) and P. carotovorum (21). Zymogram analysis of the preparation separated by isoelectric focusing showed that proteolytic activity was associated with the Coomassie blue-stained band (Fig.…”

Identification and Characterization of Hemolysin-Like Proteins Similar to RTX Toxin in Pasteurella pneumotropica

“…A 100-kDa sequence of Ca 2ϩ -dependent bacteriocin that was involved in the consensus RTX sequence in Rhizobium leguminosarum was encoded by a single gene that was unlinked to any of the activity regulator genes (30). The RTX-like metalloprotease produced by Erwinia carotovora also was encoded by the prt1 gene alone, and the expression of prt1 itself, in recombinant E. coli cells, showed a high level of protease activity (20). In both PnxIA and PnxIIA, putative acylation sites that were found in strongly cytotoxic types of RTX toxins (36) were found not to be involved.…”

Role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms

“…One of these proteinases, extracted from Erwinia carotovora subsp. carotovora, is similar in its properties to thermolysin from Bacillus thermoproteolyticus [32]. BIOCHEMISTRY (Moscow) Vol.…”