Identification of a new phospholipase C activity by analysis of an insertional mutation in the hemolytic phospholipase C structural gene of Pseudomonas aeruginosa.
The phospholipase C (PLC) gene of Pseudomonas aeruginosa encodes a heat-labile secreted hemolysin which is part of a Pi-regulated operon. The structural gene for PLC, plcS, was mutated in vitro by insertion of a tetracycline resistance gene cartridge. Gene replacement techniques were used to introduce the mutated plcS gene into the P. aeruginosa chromosome in place of the wild-type gene. The precise replacement of wild-type sequences by mutant sequences was confirmed by Southern hybridization. The mutant strain, designated PLC S, is nonhemolytic and lacks a 78-kilodalton protein corresponding to the size of the wild-type PLC. However, there is an additional phospholipase activity present in PLC S capable of hydrolyzing p-nitrophenylphosphorylcholine, a synthetic PLC substrate, and phosphatidylcholine. This enzymatic activity is not a result of a truncated product produced from the mutated plcS gene. The phospholipase activity of PLC S was identified as a nonhemolytic PLC.
Ostroff
R M
RM
Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver 80262.
Vasil
M L
ML
D002855:Chromatography, Thin Layer; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis; D004274:DNA, Recombinant; D005796:Genes; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics*; D006868:Hydrolysis; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009876:Operon; D010713:Phosphatidylcholines / Q000378:metabolism; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
C033523:4-nitrophenylphosphorylcholine; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D006460:Hemolysin Proteins; D010713:Phosphatidylcholines; D010767:Phosphorylcholine; D004262:DNA Restriction Enzymes; D010738:Type C Phospholipases