Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli.
Pseudomonas virulence is thought to depend on multiple characteristics, including the production of an extracellular alkaline protease. We report the isolation, from a PAO1 DNA genomic bank, of a cosmid carrying the structural gene coding for alkaline protease. By in vivo mutagenesis using transposon Tn1735, which functions as a transposable promoter, the expression of an 8.8-kilobase DNA fragment under control the tac promoter was obtained. When expressed in Escherichia coli, active alkaline protease was synthesized and secreted to the extracellular medium in the absence of cell lysis.
Guzzo
J
J
Laboratoire de Chimie Bactérienne, Centre National de la Recherche Scientifique, Marseille, France.
Murgier
M
M
Filloux
A
A
Lazdunski
A
A
D003001:Cloning, Molecular / Q000379:methods; D004251:DNA Transposable Elements; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005838:Genotype; D007700:Kinetics; D009154:Mutation; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012697:Serine Endopeptidases; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D010450:Endopeptidases; D012697:Serine Endopeptidases; C032923:microbial serine proteinases