Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene.
The elastase structural gene (lasB) from Pseudomonas aeruginosa PAO1 has been previously cloned on an 8-kilobase (kb) DNA fragment. The lasB gene, cloned in both orientations in pUC18, produced elastase in Escherichia coli, indicating that its promoter and translation initiation sites are functional in E. coli. Deletion analysis further defined the location of the lasB gene to a 3.0-kb EcoRI-KpnI fragment (pRB1803). Elastase prepared from E. coli TB1 (pRB1803) corresponded in molecular weight to mature P. aeruginosa extracellular elastase (33,000). The lasB gene directed the synthesis of 54- and 50-kilodalton (kDa) proteins in a bacterial cell-free transcription-translation system. The 33-, 50-, and 54-kDa proteins reacted with elastase-specific antiserum. To further characterize the lasB gene, the nucleotide sequence of the 3.0-kb EcoRI-KpnI fragment was determined. This DNA fragment contained a 1,491-base-pair open reading frame encoding 498 amino acids, corresponding to a predicted molecular weight of 53,600. The deduced amino acid sequence contained a putative signal sequence followed by a large polypeptide which preceded the mature 33-kDa elastase protein. Three zinc ligands and an active site were predicted for the mature elastase on the basis of its amino acid sequence homology with Bacillus thermoproteolyticus thermolysin.
Bever
R A
RA
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, New York 14642.
Iglewski
B H
BH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D005786:Gene Expression Regulation; D005796:Genes; D005798:Genes, Bacterial; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D015032:Zinc / Q000378:metabolism
D004269:DNA, Bacterial; D008024:Ligands; D021382:Protein Sorting Signals; D004262:DNA Restriction Enzymes; D010196:Pancreatic Elastase; D015032:Zinc