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PMID: 6401709 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Cloning, and expression in Escherichia coli K-12, of the chromosomal hemolysin (phospholipase C) determinant of Pseudomonas aeruginosa.

Journal of bacteriology ·Vol. 153 ·No. 2 ·1983-02-00 ·Pages 909-15

Coleman K, Dougan G, Arbuthnott JP

Abstract

A hemolysin determinant was cloned from Pseudomonas aeruginosa PA103 by inserting Sau3a-generated DNA fragments between the BamHI sites of the lambda replacement vector WL47.1. A 9.5-kilobase HindIII fragment encoding the hemolysin was subcloned from this phage and inserted into the plasmid vector pHC79 to generate the recombinant plasmid pKC95. Escherichia coli K-12 strains harboring pKC95 exhibited zones of hemolysis after several days of growth on blood agar plates. Hemolysis was shown to be due to phospholipase C activity by using the chromogenic substrate p-nitrophenylphosphorylcholine. Deletion mutants of pKC95 were isolated, and polypeptides expressed from these plasmids were examined by using the E. coli minicell system. A polypeptide of 78,000 daltons was associated with phospholipase C activity. The hemolytic activity was cell associated when expressed in E. coli.

MeSH Terms
Cloning, Molecular Escherichia coli/enzymology,genetics Genes, Bacterial Hemolysin Proteins/genetics Hemolysis Phospholipases/genetics Plasmids Pseudomonas aeruginosa/enzymology,genetics Type C Phospholipases/genetics,metabolism
Chemicals
Hemolysin Proteins Phospholipases Type C Phospholipases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Coleman K
Dougan G
Arbuthnott J P
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23 references, click to expand
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.153.2.909-915.1983
Published
1983-02-00
Pages
909-15
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC221713
Subset
IM
External Links
PubMed source DOI: 10.1128/jb.153.2.909-915.1983 PMC Full Text View journal details
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