Protein binding sites involved in the assembly of the KplE1 prophage intasome

Abstract

The organization of the recombination regions of the KplE1 prophage in Escherichia coli K12 differs from that observed in the lambda prophage. Indeed, the binding sites characterized for the IntS integrase, the TorI recombination directionality factor (RDF) and the integration host factor (IHF) vary in number, spacing and orientation on the attL and attR regions. In this paper, we performed site-directed mutagenesis of the recombination sites to decipher if all sites are essential for the site-specific recombination reaction and how the KplE1 intasome is assembled. We also show that TorI and IntS form oligomers that are stabilized in the presence of their target DNA. Moreover, we found that IHF is the only nucleoid associated protein (NAP) involved in KplE1 recombination, although it is dispensable. This is consistent with the presence of only one functional IHF site on attR and none on attL.

DnaJ (Hsp40 Protein) Binding to Folded Substrate Impacts KplE1 Prophage Excision Efficiency

“…1). In the presence of BMH, immunoblot analysis showed a characteristic oligomerization of TorI when incubated alone (29). This was seen by the presence of bands corresponding to dimeric through to hexameric forms of TorI (Fig.…”

Bacteriophage mv4 Site-Specific Recombination: the Central Role of the P2 mv4 Int-Binding Site

“…In well-characterized site-specific recombination systems, the protein binding sites at the att recombination sites are not all used in the same way for integrative or excisive recombination (1)(2)(3). The integrase of phage is a heterobivalent recombinase that binds two different families of DNA sequences: the "arm-type" and the "core-type" binding sites (4).…”

“…The overall organization of the mv4 attP site highlights the absence of binding sites for bending proteins between the Int armtype binding sites and the core region (8); this is unlike what has been found for the attP site in well-characterized phages (3,(10)(11)(12)(13). Analysis of the core region sequence does not reveal any Int core-type binding sites (14), and the Int P2 arm-binding site has a noticeable position close to the left border of the core region (Fig.…”

Transcription termination controls prophage maintenance in Escherichia coli genomes

“…KplE1 (also named CPS-53) is highly defective (it has only a 10-kb genome). However, it is fully competent for site-specific recombination and provides fitness to its host under oxidative stress conditions (28)(29)(30). HK620 is a fully infectious P22-like phage that shares a highly conserved site-specific recombination module with KplE1 (27,28).…”