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Export of protein in Escherichia coli: a novel mutation in ompC affects expression of other major outer membrane
Abstract:
A mutation within the ompC structural gene of Escherichia coli K-12 which affects expression of outer membrane proteins was characterized. The mutation consisted of a 6-base-pair deletion near the 3' end of the gene which removed the amino acids Val-300 and Gly-301 of the mature coding sequence but otherwise left the reading frame intact. The deletion occurred within a region highly conserved among the porins. No protein product was detected from a single copy of the mutant gene. The mutation caused a trans-dominant decrease in the expression of a wild-type ompC allele. The mutation caused a similar decrease in the amounts of OmpA, OmpF, LamB, and Lc proteins, yet it did not appear to affect the minor outer membrane proteins. It had no significant effect on transcription from either ompF or ompC promoters as measured with lacZ operon fusions. The effects of the mutation on other proteins were completely eliminated when the signal sequence was disrupted so that the mutant protein no longer interacted with the secretion machinery of the cell but instead accumulated as precursor in the cytoplasm. A model is proposed involving the translocation of proteins to the outer membrane and the importance of protein conformation in this process.
Insights
A specific mutation in the Escherichia coli ompC gene disrupts outer membrane protein production. This finding suggests protein conformation is crucial for translocation to the cell surface.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Escherichia coli utilizes outer membrane proteins (OMPs) for various cellular functions.
- The ompC gene encodes a major porin protein essential for nutrient uptake and cell envelope integrity.
- Understanding mutations affecting OMP expression is key to deciphering bacterial physiology and pathogenesis.
Purpose of the Study:
- To characterize a specific mutation within the ompC gene of Escherichia coli K-12.
- To investigate the mutation's impact on the expression of OMPs and related proteins.
- To elucidate the role of protein conformation in the translocation of OMPs.
Main Methods:
- Site-directed mutagenesis to introduce a 6-base-pair deletion in the ompC gene.
- Analysis of protein expression using techniques like SDS-PAGE and Western blotting.
- Assessment of transcriptional activity using lacZ operon fusions.
Main Results:
- A 6-bp deletion in ompC removed Val-300 and Gly-301, abolishing detectable protein product.
- The mutation exhibited trans-dominant negative effects, decreasing expression of wild-type OmpC and other OMPs (OmpA, OmpF, LamB, Lc).
- Transcriptional activity of ompC and ompF promoters remained unaffected, indicating post-transcriptional regulation.
Conclusions:
- The ompC mutation disrupts OMP translocation, likely due to altered protein conformation.
- The signal sequence is critical for mediating the interaction of the mutant protein with the secretion machinery.
- Protein conformation plays a vital role in the proper transport and insertion of OMPs into the bacterial outer membrane.