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Published on: November 12, 2012
Escherichia coli K-12 outer membrane protein (OmpA) as a bacteriophage receptor: analysis of mutant genes expressing
Abstract:
The outer membrane protein OmpA of Escherichia coli K-12 serves as a receptor for a number of T-even-like phages. We have isolated a series of ompA mutants which are resistant to such phages but which still produce the OmpA protein. None of the mutants was able to either irreversibly or reversibly bind the phage with which they had been selected. Also, the OmpA protein is required for the action of colicins K and L and for the stabilization of mating aggregates in conjugation. Conjugal proficiency was unaltered in all cases. Various degrees of colicin resistance was found; however, the resistance pattern did not correlate with the phage resistance pattern. DNA sequence analyses revealed that, in the mutants, the 325-residue OmpA protein had suffered the following alterations: Gly-65----Asp, Gly-65----Arg, Glu-68----Gly, Glu-68----Lys (two isolates), Gly-70----Asp (four isolates), Gly-70----Val, Ala-Asp-Thr-Lys-107----Ala-Lys (caused by a 6-base-pair deletion), Val-110----Asp, and Gly-154----Ser. These mutants exhibited a complex pattern of resistance-sensitivity to 14 different OmpA-specific phages, suggesting that they recognize different areas of the protein. In addition to the three clusters of mutational alterations around residues 68, 110, and 154, a site around residue 25 has been predicted to be involved in conjugation and in binding of a phage and a bacteriocin (R. Freudl, and S. T. Cole, Eur. J. Biochem, 134:497-502, 1983; G. Braun and S. T. Cole, Mol. Gen. Genet, in press). These four areas are regularly spaced, being about 40 residues apart from each other. A model is suggested in which the OmpA polypeptide repeatedly traverses the outer membrane in cross-beta structure, exposing the four areas to the outside.
Insights
Mutations in the outer membrane protein OmpA of Escherichia coli K-12 confer resistance to phages and colicins. These ompA mutants reveal specific regions of OmpA involved in phage binding and colicin activity.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- The outer membrane protein OmpA in Escherichia coli K-12 is a crucial receptor for T-even-like phages.
- OmpA also plays a role in the activity of colicins K and L and in stabilizing mating aggregates during bacterial conjugation.
Purpose of the Study:
- To investigate the functional domains of OmpA by isolating and characterizing mutants resistant to specific phages.
- To understand the relationship between OmpA structure, phage binding, colicin resistance, and conjugation proficiency.
Main Methods:
- Isolation of ompA mutants exhibiting resistance to T-even-like phages.
- Phenotypic analysis of mutants for phage binding, colicin sensitivity, and conjugal proficiency.
- DNA sequencing to identify mutations within the ompA gene and corresponding amino acid alterations.
Main Results:
- Isolated mutants remained capable of producing OmpA but lost phage binding ability.
- Mutations occurred at specific residues (e.g., 65, 68, 70, 107, 110, 154), affecting phage resistance in complex patterns.
- Colicin resistance varied among mutants and did not correlate with phage resistance patterns.
- Conjugal proficiency was unaffected in all isolated mutants.
Conclusions:
- Specific regions of the OmpA protein are critical for phage receptor function.
- OmpA's role in colicin activity is distinct from its role in phage binding.
- The findings support a model where OmpA traverses the outer membrane, exposing functionally important regions at regular intervals.
