Control and Regulation of KplE1 Prophage Site-specific Recombination

Abstract

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

Pathogenicity Island Cross Talk Mediated by Recombination Directionality Factors Facilitates Excision from the Chromosome

“…None of the 35 identified ORFs yielded any similarity to previously described excisionases. However, when the vpiT (VC0817) amino acid sequence was submitted to HHpred, 52 amino acids near the N terminus displayed a distant relationship to TorI, a previously characterized excisionase (25,31,52). Although the shared identity in this region was 12%, with an E value of 0.0061, the predicted secondary elements of these regions were nearly identical (see Fig.…”

“…In TR integrase-encoding prophages, it was demonstrated that the excision event also requires an additional protein, an excisionase or recombination directionality factor (RDF) (24)(25)(26)(27)(28). In fact, it is believed that all TR integrases require an RDF to perform excision (20,29).…”

Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism

“…Examination of the deposited genome of E. coli DH10b (the parent of Top10) (GenBank TM accession number NC_010473.1) revealed a three-ORF cluster with high sequence identity to S. flexneri bacteriophage (gtr) glucosylation clusters, belonging to the prophage KplE1 (19). The -red recombination system was used to remove the first gene in this cluster, a gtrA homologue annotated yfdG, which overlaps with the first four nucleotides of the gtrB homologue, yfdH.…”

“…The association of glucosylation modification with the Wzy-dependent pathway may reflect the limits to which systems have been characterized at a bioinformatics and biochemical level or may result from the glycosylation system being critically dependent on elements of the Wzy-dependent machinery. Here, we examine the possibility that the ABC transporter-dependent pathway provides an intrinsic mechanistic barrier for the glucosylation process by examining the potential for the native E. coli K-12 glucosylation machinery encoded by prophage KplE1 (19) to modify target residues supplied by a heterologous ABC transporter-dependent O-antigen from Raoultella terrigena ATCC 33257 (see Fig. 2).…”

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).…”