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Abstract:
Colicin M (Cma) is a protein toxin that is formed by E. coli strains that carry ColBM plasmids. It is imported into the periplasm of sensitive cells via a receptor-dependent energy-coupled process. It kills E. coli cells by inhibition of murein (peptidoglycan) precursor incorporation into the existing murein in that it cleaves the phosphate ester bond between the precursor and the lipid carrier that translocates the precursor across the cytoplasmic membrane. The resulting C 55 polyisoprenol no longer enters the reaction cycle, murein synthesis stops and cells lyse. E. coli cells that synthesize Cma are protected by an immunity protein, Cmi, which in the periplasm inactivates Cma. E. coli mutants which are resistant to Cma carry mutations in genes, fhuA, tonB, exbB, exbD, which are involved in Cma import from the outside into the periplasm. We recently found that an additional type of Cma resistant mutant carries a mutation in fkpA that encodes a periplasmic prolyl cis-trans isomerase (PPIase) / chaperone. Spontaneous fkpA deletion and point mutants in the PPIase domain are completely resistant to high titers (10 5 ) of Cma. The crystal structure of Cma reveals a compact form that must unfold during translocation across the outer membrane. It is assumed that this involves a trans-to-cis prolyl isomerisation of Cma that is converted back to trans upon refolding in the periplasm. Cma refolding is catalysed by FkpA. Regardless whether Cma is imported or secreted with a fused signal sequence into the periplasm, it requires FkpA to be active. To identify the residue that might be cis-trans isomerized, the 15 proline residues were individually replaced by alanine. The mutant Cma’s were fully active except three which displayed 1% activity. Two of them are not imported. The one that remains inactive in the periplasm has a crystal structure identical to wild-type Cma which makes it unlikely that the mutation changes the phosphatase active center that is located far from the proline residue. It is proposed that the proline residue of the inactive imported mutant is targeted by FkpA. Sequence and structure of the phosphatase domain of Cma is unique. The active center was therefore mapped by random and site-specific mutagenesis. The mutations center in a surface-exposed region. An aspartate residue was defined as a likely catalytic site since conversion to asparagine or glutamate abolishes Cma activity. The residues implicated in phosphatase catalysis are highly conserved in Cma-like proteins of other species than E. coli.
