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Characteristics of detergent-resistant phospholipase A overproduced in E. coli cells bearing its cloned structural
Abstract:
Detergent-resistant phospholipase A (DR-phospholipase A) of E. coli is a 28K-dalton protein and is exclusively located in the outer membrane. We cloned the pldA gene of E. coli, which is responsible for the activity of DR-phospholipase A. Strains bearing the plasmid which contained the pldA gene yielded a large amount of the outer membrane protein with a molecular weight of about 28K daltons and overproduced 20 to 65 times as much DR-phospholipase A activity as the wild type strain. Experiments with minicells and maxicells revealed that the pldA-containing plasmid was coding for a 28K protein. These results strongly indicated that pldA is the structural gene for DR-phospholipase A. There was apparently no difference with respect to the association of the enzyme with the envelope fraction between the overproducer and the wild type strain. The overproduced enzyme was properly transported to the outer membrane. Neither the growth rate nor the phospholipid composition of the overproducer was remarkably different from in the wild type strain. Thus, the overproduction of DR-phospholipase A apparently caused no phenotypic variations. E. coli has very excessive ability to transport and integrate the outer membrane protein.
Insights
Researchers cloned the E. coli pldA gene, confirming it codes for detergent-resistant phospholipase A (DR-phospholipase A). Overproducing this outer membrane protein caused no significant changes in cell growth or composition.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- E. coli outer membrane contains detergent-resistant phospholipase A (DR-phospholipase A), a 28-kDa protein.
- The specific gene responsible for DR-phospholipase A activity was previously unidentified.
Purpose of the Study:
- To identify and clone the gene encoding DR-phospholipase A in E. coli.
- To characterize the overproduction of DR-phospholipase A and its effects on the cell.
Main Methods:
- Cloning of the pldA gene from E. coli.
- Expression studies using plasmids in E. coli strains.
- Analysis of protein production and enzyme activity in minicells and maxicells.
- Assessment of cellular growth rate and phospholipid composition.
Main Results:
- The pldA gene was successfully cloned and confirmed to encode the 28-kDa DR-phospholipase A.
- E. coli strains carrying the pldA-containing plasmid overproduced DR-phospholipase A activity 20-65 fold compared to wild type.
- Overproduced DR-phospholipase A was correctly localized to the outer membrane.
- No significant differences in growth rate or phospholipid composition were observed between overproducing and wild-type strains.
Conclusions:
- The pldA gene is the structural gene for E. coli DR-phospholipase A.
- E. coli exhibits a high capacity for transporting and integrating outer membrane proteins.
- Overproduction of DR-phospholipase A does not induce significant phenotypic variations in E. coli.