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Sensitivity of Escherichia coli to cloacin DF13 involves the major outer membrane protein OmpF
1Department of Genetics, University of Leicester, England.
Abstract:
Fourteen spontaneous cloacin DF13-insensitive mutants of an Escherichia coli strain expressing the aerobactin-cloacin DF13 receptor protein IutA were isolated. The mutants fell into three classes on the basis of outer membrane profiles analyzed by electrophoresis in denaturing polyacrylamide gels. The most frequent class lacked the IutA protein and was unable to bind cloacin DF13 or aerobactin. A second class of mutants had lost protein species corresponding in size to the porin proteins OmpF and OmpC. To determine which porin was required for the bactericidal activity of cloacin DF13, defined strains with mutations at the ompB (ompR envZ) locus were transformed with a recombinant plasmid carrying the iutA gene and screened for cloacin DF13 sensitivity. OmpF- strains, whether OmpC+ or OmpC-, were insensitive to cloacin DF13, indicating involvement of the OmpF protein in cloacin DF13 killing. An OmpC- OmpF+ strain, on the other hand, was more sensitive than the wild-type parent strain, probably because of compensatory overexpression of OmpF. The third class of cloacin DF13-insensitive mutant had lost an outer membrane protein of approximately 31 kDa. The nature and function of this protein are not yet known, but it is not the protease OmpT. Mutants of classes 2 and 3 bound cloacin DF13 and aerobactin as effectively as the cloacin DF13-sensitive parental strain, indicating that they remained IutA+. We propose that these mutants (more accurately described as cloacin DF13 tolerant) are defective in translocation of the active portion of cloacin DF13 across the bacterial membranes.
Insights
Mutations affecting the OmpF porin in Escherichia coli confer resistance to cloacin DF13 by preventing its entry into the bacterial cell. This study identifies OmpF as crucial for cloacin DF13 activity.
Area of Science:
- Microbiology
- Bacterial Physiology
- Outer Membrane Proteins
Background:
- Cloacin DF13 is a bacteriocin that targets Escherichia coli.
- The aerobactin-cloacin DF13 receptor protein, IutA, facilitates cloacin DF13 binding.
- Outer membrane proteins play critical roles in bacterial interactions with external agents.
Purpose of the Study:
- To investigate the genetic basis of spontaneous cloacin DF13 resistance in Escherichia coli.
- To identify outer membrane proteins involved in cloacin DF13 sensitivity and uptake.
- To elucidate the mechanism by which cloacin DF13 exerts its bactericidal effect.
Main Methods:
- Isolation and characterization of spontaneous cloacin DF13-insensitive mutants.
- Outer membrane protein profiling using denaturing polyacrylamide gel electrophoresis.
- Genetic manipulation of porin genes (ompF, ompC) and screening for cloacin DF13 sensitivity.
- Transformation of defined mutant strains with a plasmid carrying the iutA gene.
Main Results:
- Mutants were classified into three groups based on outer membrane profiles.
- The most common class lacked the IutA receptor, rendering them insensitive to cloacin DF13 and aerobactin.
- A significant class of mutants lost OmpF and/or OmpC porins; OmpF deficiency conferred cloacin DF13 insensitivity.
- A third class lost an unknown ~31 kDa outer membrane protein, but retained IutA and aerobactin binding.
Conclusions:
- The OmpF porin is essential for the bactericidal activity of cloacin DF13 in Escherichia coli.
- Mutations affecting OmpF or potentially a novel 31 kDa outer membrane protein can lead to cloacin DF13 tolerance.
- Insensitivity in mutants lacking OmpF or the unknown protein suggests a defect in the translocation of cloacin DF13 across bacterial membranes, rather than initial binding.