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Identification and characterization of the Escherichia coli envC gene encoding a periplasmic coiled-coil protein with
Hiroshi Hara1, Setsuko Narita, Doris Karibian
1Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, Japan. hhara@molbiol.saitama-u.ac.jp
Abstract:
PM61 is a chain-forming envC strain of Escherichia coli with a leaky outer membrane. It was found to have an oversized penicillin-binding protein 3, which was the result of an IS4 insertion in the prc gene. The other properties of PM61 were caused by the envC mutation. We cloned the envC (yibP) gene and identified the mutation site, causing a single residue substitution, H366Y, in the PM61 envC allele. The gene product was predicted to be a periplasmic protein having coiled-coil structure in the N-terminal region and homology to lysostaphin in the C-terminal region. Overexpression of envC inhibited cell growth, and overexpression of the PM61 mutant allele caused cell lysis. Disruption of the chromosomal envC caused the same defects as the envC point mutation, indicating the gene is dispensable for growth but important for normal septation/separation and cell envelope integrity.
Insights
The envC gene in Escherichia coli is crucial for cell division and envelope integrity. Mutations in envC lead to chain formation and outer membrane defects, impacting bacterial morphology and survival.
Area of Science:
- Microbiology
- Molecular Biology
- Cell Biology
Background:
- The Escherichia coli strain PM61 exhibits chain-forming morphology and a leaky outer membrane.
- PM61 possesses an enlarged penicillin-binding protein 3 due to IS4 insertion in the prc gene.
- The observed phenotypes of PM61 are primarily attributed to a mutation in the envC gene.
Purpose of the Study:
- To clone and characterize the envC (yibP) gene from Escherichia coli.
- To identify the specific mutation within the envC gene responsible for the PM61 phenotype.
- To elucidate the role of envC in bacterial cell division, cell envelope integrity, and morphology.
Main Methods:
- Cloning of the envC (yibP) gene.
- Site-directed mutagenesis to identify the mutation site (H366Y).
- Gene disruption and overexpression studies in Escherichia coli.
Main Results:
- The envC mutation in PM61 was identified as a single residue substitution, H366Y.
- The envC gene product is predicted to be a periplasmic protein with specific structural domains.
- Overexpression of envC inhibited growth, while overexpression of the mutant allele induced cell lysis.
- Disruption of the chromosomal envC gene recapitulated the observed defects.
Conclusions:
- The envC gene is essential for proper cell septation, separation, and maintaining cell envelope integrity in Escherichia coli.
- While dispensable for growth, envC plays a critical role in bacterial morphology and cell division processes.
- The H366Y mutation in envC significantly impacts Escherichia coli cell envelope and division.