Abstract
Temperate phages mediate gene transfer and can modify the properties of their host organisms through the acquisition of novel genes, a process called lysogeny. The KplE1 prophage is one of the 10 prophage regions in Escherichia coli K12 MG1655. KplE1 is defective for lysis but fully competent for site-specific recombination. The TorI recombination directionality factor is strictly required for prophage excision from the host genome. We have previously shown that DnaJ promotes KplE1 excision by increasing the affinity of TorI for its site-specific recombination DNA target. Here, we provide evidence of a direct association between TorI and DnaJ using in vitro cross-linking assays and limited proteolysis experiments that show that this interaction allows both proteins to be transiently protected from trypsin digestion. Interestingly, NMR titration experiments showed that binding of DnaJ involves specific regions of the TorI structure. These regions, mainly composed of α-helices, are located on a surface opposite the DNA-binding site. Taken together, we propose that DnaJ, without the aid of DnaK/GrpE, is capable of increasing the efficiency of KplE1 excision by causing a conformational stabilization that allows TorI to adopt a more favorable conformation for binding to its specific DNA target.
Highlights
DnaJ positively modulates KplE1 prophage excision and is involved in lysogeny escape
We have previously shown that DnaJ promotes KplE1 excision by increasing the affinity of TorI for its site-specific recombination DNA target
Direct Association of DnaJ and TorI—We have previously shown that DnaJ has the ability to increase the excision efficiency of the KplE1 prophage from the K12 bacterial chromosome [24]
Summary
DnaJ positively modulates KplE1 prophage excision and is involved in lysogeny escape. Results: The recombination directionality factor TorI, from KplE1 prophage, interacts with the DnaJ chaperone, and they protect each other from limited trypsin digestion. NMR titration experiments showed that binding of DnaJ involves specific regions of the TorI structure. These regions, mainly composed of ␣-helices, are located on a surface opposite the DNA-binding site. We propose that DnaJ, without the aid of DnaK/GrpE, is capable of increasing the efficiency of KplE1 excision by causing a conformational stabilization that allows TorI to adopt a more favorable conformation for binding to its specific DNA target. Site-specific recombination is used by temperate phages to integrate and excise from their host chromosome [1]. Classical functions of the DnaK/ DnaJ/GrpE machinery under low stress conditions include folding of newly synthesized proteins, protein assembly and disassembly, and translocation of proteins into organelles (15, The abbreviations used are: RDF, recombination directionality factor; BMH, bismaleimidohexane; IHF, integration host factor; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
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