TorI, a response regulator inhibitor of phage origin in Escherichia coli

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KplE1 is one of the 10 prophage regions of Escherichia coli K12, located at 2464 kb on the chromosome. KplE1 is defective for lysis, but it is fully competent for excisive recombination. In this study, we have mapped the binding sites of the recombination proteins, namely IntS, TorI, and IHF on attL and attR, and the organization of these sites suggests that the intasome is architecturally different from the canonical form. We also measured the relative contribution of these proteins to both excisive and integrative recombination by using a quantitative in vitro assay. These experiments show a requirement of the TorI excisionase for excisive recombination and of the IntS integrase for both integration and excision. Moreover, we observed a strong influence of the supercoiled state of the substrates. The KplE1 recombination module, composed of the integrase and excisionase genes together with the attL and attR DNA regions, is highly similar to that of several phages infecting various E. coli strains as well as Shigella flexneri and Shigella sonnei. The in vitro recombination data reveal that HK620 and KplE1 att sequences are exchangeable. This study thus defines a new sitespecific recombination module, and implications for the mechanism and regulation of recombination are discussed.Phage has long served as a model system for studies of regulated site-specific recombination (1). Indeed, bacteriophages such as may choose between the lytic and the lysogenic cycle for their propagation in the bacterial host (2). In conditions favorable for bacterial growth the phage genome is inserted into the host genome by an integrative recombination reaction, which takes place between DNA attachment sites called attP and attB, in the phage and the bacterial genome, respectively. As a result, the integrated DNA (or prophage DNA) is bounded by hybrid attachment sites, termed attL and attR. In response to a change in the physiological state of the bacteria, mainly in response to stress conditions such as DNA damage, phage DNA is excised from the host chromosome. This excisive reaction recombines attL and attR to restore the attP and attB sites on the circular and Escherichia coli

“…Protein Purifications-IHF and TorI proteins were overproduced and purified as described (23,29). IntS was produced from C41(DE3) harboring plasmid pETintS.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Control and Regulation of KplE1 Prophage Site-specific Recombination

Panis¹,

Méjean

Ansaldi

2007

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“…Immunoprecipitation-The E. coli ⌬ 3 strain harboring the pGPJ-His plasmid (30) was transformed with pBTorI (15). Cells were grown until A 600 reached 0.5 unit.…”

Section: Volume 286 • Number 45 • November 11 2011mentioning

confidence: 99%

“…The torI gene is the last gene of the KplE1 prophage genome and was originally identified using a genetic multicopy approach as a negative regulator of the torCAD operon that encodes the trimethylamine-oxide reductase respiratory system in E. coli (15). Despite its role as an inhibitor of the TorR response regulator, later work has shown that TorI is in fact a bona fide RDF for KplE1 phage excision (10).…”

mentioning

confidence: 99%

Chaperone-assisted Excisive Recombination, a Solitary Role for DnaJ (Hsp40) Chaperone in Lysogeny Escape

Champ¹,

Puvirajesinghe²,

Perrody³

et al. 2011

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“…Some RDFs, such as Cox proteins of phages P2 and HP1, have additional regulatory functions outside recombination (9). TorI itself was originally identified as a negative response regulator of the TorR protein, which activates transcription of the torCAD operon, encoding the trimethylamine-N-oxide reductase respiratory system (10).…”

mentioning

confidence: 99%

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

Puvirajesinghe

Elantak

Lignon

et al. 2012

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Background: 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. Conclusion: DnaJ stabilizes TorI recombination directionality factor conformation. Significance: DnaJ cochaperone can bind folded substrates and induces the conformational stabilization of the TorI protein.