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PMID: 2120196 Published · ppublish English Comparative Study Journal Article Research Support, U.S. Gov't, P.H.S.

Molecular comparison of a nonhemolytic and a hemolytic phospholipase C from Pseudomonas aeruginosa.

Journal of bacteriology ·Vol. 172 ·No. 10 ·1990-10-00 ·Pages 5915-23

Ostroff RM, Vasil AI, Vasil ML

Abstract

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.

MeSH Terms
Amino Acid Sequence Base Sequence Blotting, Southern Cloning, Molecular/methods DNA, Bacterial/genetics,isolation & purification Escherichia coli/genetics Genotype Hemolysis Isoenzymes/genetics,metabolism Molecular Sequence Data Mutation Phenotype Plasmids Pseudomonas aeruginosa/enzymology,genetics Recombinant Proteins/metabolism Restriction Mapping Sequence Homology, Nucleic Acid Substrate Specificity Type C Phospholipases/genetics,metabolism
Chemicals
DNA, Bacterial Isoenzymes Recombinant Proteins Type C Phospholipases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Ostroff R M
Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver 80262.
Vasil A I
Vasil M L
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.172.10.5915-5923.1990
Published
1990-10-00
Pages
5915-23
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC526912
Subset
IM
Grants
NIAID NIH HHS · AI15940 · United States
Databases
GENBANK
M59304
External Links
PubMed source DOI: 10.1128/jb.172.10.5915-5923.1990 PMC Full Text View journal details
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