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Identification of a Dps contamination in Mitomycin-C-induced expression of Colicin Ia
Joka Pipercevic1, Roman P Jakob1, Ricardo D Righetto2
1Biozentrum, University of Basel, 4056 Basel, Switzerland.
Abstract:
Colicins are bacterial toxins targeting Gram-negative bacteria, including E. coli and related Enterobacteriaceae strains. Some colicins form ion-gated pores in the inner membrane of attacked bacteria that are lethal to their target. Colicin Ia was the first pore-forming E. coli toxin, for which a high-resolution structure of the monomeric full-length protein was determined. It is so far also the only colicin, for which a low-resolution structure of its membrane-inserted pore was reported by negative-stain electron microscopy. Resolving this structure at the atomic level would allow an understanding of the mechanism of toxin pore formation. Here, we report an observation that we made during an attempt to determine the Colicin Ia pore structure at atomic resolution. Colicin Ia was natively expressed by mitomycin-C induction under a native SOS promotor and purified following published protocols. The visual appearance in the electron microscope of negatively stained preparations and the lattice parameters of 2D crystals obtained from the material were highly similar to those reported earlier resulting from the same purification protocol. However, a higher-resolution structural analysis revealed that the protein is Dps (DNA-binding protein from starved cells), a dodecameric E. coli protein. This finding suggests that the previously reported low-resolution structure of a "Colicin Ia oligomeric pore" actually shows Dps.
Insights
Researchers attempted to determine the atomic structure of the Colicin Ia pore but discovered it was actually the DNA-binding protein from starved cells (Dps). This finding reinterprets previous low-resolution structural data of bacterial toxin pores.
Area of Science:
- Structural biology
- Bacteriology
- Molecular toxicology
Background:
- Colicins are protein toxins produced by E. coli and other bacteria that target Gram-negative bacteria.
- Some colicins form lethal ion pores in the bacterial inner membrane.
- Colicin Ia is a well-studied pore-forming toxin, with its monomeric structure resolved, but its pore structure remains poorly understood.
Purpose of the Study:
- To determine the high-resolution atomic structure of the membrane-inserted Colicin Ia pore.
- To elucidate the mechanism of Colicin Ia pore formation.
Main Methods:
- Native expression of Colicin Ia in E. coli.
- Purification of the protein using established protocols.
- Negative-stain electron microscopy and 2D crystallization.
- Higher-resolution structural analysis of the purified protein and crystals.
Main Results:
- The purified protein, intended to be Colicin Ia, exhibited characteristics similar to previously reported preparations.
- Higher-resolution structural analysis revealed the protein to be Dps (DNA-binding protein from starved cells), a dodecameric E. coli protein.
- The previously reported low-resolution structure of a 'Colicin Ia oligomeric pore' is likely Dps.
Conclusions:
- The protein previously identified as Colicin Ia in pore studies is likely Dps.
- This finding necessitates a re-evaluation of existing structural data on Colicin Ia pores.
- Further studies are required to determine the true structure and mechanism of the Colicin Ia pore.
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