Molecular comparison of a nonhemolytic and a hemolytic phospholipase C from Pseudomonas aeruginosa.
Pseudomonas aeruginosa produces two secreted phospholipase C (PLC) enzymes. The expression of both PLCs is regulated by Pi. One of the PLCs is hemolytic, and one is nonhemolytic. Low-stringency hybridization studies suggested that the genes encoding these two PLCs shared DNA homology. This information was used to clone plcN, the gene encoding the 77-kilodalton nonhemolytic PLC, PLC-N. A fragment of plcN was used to mutate the chromosomal copy of plcN by the generation of a gene interruption mutation. This mutant produces 55% less total PLC activity than the wild type, confirming the successful cloning of plcN. plcN was sequenced and encodes a protein which is 40% identical to the hemolytic PLC (PLC-H). The majority of the homology lies within the NH2 two-thirds of the proteins, while the remaining third of the amino acid sequence of the two proteins shows very little homology. Both PLCs hydrolyze phosphatidylcholine; however, each enzyme has a distinct substrate specificity. PLC-H hydrolyzes sphingomyelin in addition to phosphatidylcholine, whereas PLC-N is active on phosphatidylserine as well as phosphatidylcholine. These studies suggest structure-function relationships between PLC activity and hemolysis.
Ostroff
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RM
Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver 80262.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005838:Genotype; D006461:Hemolysis; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D007527:Isoenzymes; D011994:Recombinant Proteins; D010738:Type C Phospholipases