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Apparent bacteriophage-binding region of an Escherichia coli K-12 outer membrane protein
Abstract:
The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli. It serves as the receptor for several T-even-like phages and is required for the action of certain colicins and for the stabilization of mating aggregates in conjugation. We have isolated two mutant alleles of the cloned ompA gene which produce a protein that no longer functions as a phage receptor. Bacteria possessing the mutant proteins were unable to bind the phages, either reversibly or irreversibly. However, both proteins still functioned in conjugation, and one of them conferred colicin L sensitivity. DNA sequence analysis showed that the phage-resistant, colicin-sensitive phenotype exhibited by one mutant was due to the amino acid substitution Gly leads to Arg at position 70. The second mutant, which contained a tandem duplication, encodes a larger product with 8 additional amino acid residues, 7 of which are a repeat of the sequence between residues 57 and 63. In contrast to the wild-type OmpA protein, this derivative was partially digested by pronase when intact cells were treated with the enzyme. The protease removed 64 NH2-terminal residues, thereby indicating that this part of the protein is exposed to the outside. It is argued that the phage receptor site is most likely situated around residues 60 to 70 of the OmpA protein and that the alterations characterized have directly affected this site.
Insights
Mutations in the OmpA protein of Escherichia coli disrupt its function as a phage receptor. These OmpA variants still support conjugation and colicin L activity, suggesting specific alterations in the phage binding site.
Area of Science:
- Molecular Biology
- Microbiology
- Protein Engineering
Background:
- OmpA is a major outer membrane protein in Escherichia coli, crucial for phage binding, colicin activity, and bacterial conjugation.
- Understanding OmpA's structural and functional domains is key to deciphering its roles in bacterial interactions.
Purpose of the Study:
- To investigate the role of specific OmpA protein regions in phage receptor function.
- To characterize mutations affecting OmpA's interaction with T-even phages.
Main Methods:
- Isolation and characterization of mutant ompA alleles.
- Bacterial phage binding assays (reversible and irreversible).
- Assays for conjugation and colicin L sensitivity.
- DNA sequencing to identify mutations.
- Pronase digestion to assess protein exposure.
Main Results:
- Two mutant ompA alleles were isolated, producing OmpA proteins non-functional as phage receptors.
- Mutant proteins retained function in conjugation and, in one case, colicin L sensitivity.
- A Gly70Arg substitution caused phage resistance and colicin sensitivity.
- A tandem duplication mutant produced a larger OmpA with altered protease susceptibility, indicating external exposure of N-terminal residues.
Conclusions:
- The phage receptor site of OmpA is likely located around residues 60-70.
- Specific amino acid alterations in this region directly impact phage binding.
- OmpA's structural integrity and external accessibility are critical for its phage receptor function.