Short sequence-paper The Shiga-toxin VT2-encoding bacteriophage B297 integrates at a distinct position in the Escherichia coli genome Henri De Greve a,b, * , Cao Qizhi a,1 , Francine Deboeck a , Jean-Pierre Hernalsteens a a Laboratorium Genetische Virologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium b Department of Immunology, Parasitology, and Ultrastructure, Flanders Interuniversity Institute for Biotechnology, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium Received 27 February 2002; received in revised form 3 September 2002; accepted 23 September 2002 Abstract The plaque-forming VT2-encoding lambdoid bacteriophage B297 was isolated from a Belgian clinical Escherichia coli O157:H7 isolate. PCR walking, starting from the int gene of phage B297, demonstrated that the B297 prophage integrated in the yecE gene of a lysogenic E. coli K12 strain. This integration site, in E. coli K12 and in the original clinical O157:H7 isolate, was confirmed by PCR using primers flanking this site. The excisionase protein of phage B297 is identical to the excisionase of VT1-encoding phage VT1-Sakai, while the integrases, which are 82% identical, show significant sequence divergence in the central and C-terminal region. This can explain the different integration sites of both prophages. The activity of the integrase was proven by its ability to mediate the integration of a suicide plasmid, carrying the attachment site of B297, at the appropriate position in the E. coli chromosome. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Shiga-toxin; VT2-encoding bacteriophage; Attachment site; Site-specific recombination; Escherichia coli O157 Enterohemorrhagic Escherichia coli (EHEC) strains cause human infections that can result in hemolytic colitis (HC) or hemolytic uremic syndrome (HUS). These symp- toms are the consequence of the action of Shiga-toxins, produced by the EHEC strains. The genes encoding the Shiga-toxins VT1 and VT2 are located on lambdoid bac- teriophages, under the control of the late gene promoter. The complete nucleotide sequences of the VT2-encoding phages 933 W [1] and VT2-Sakai [2], as well as the VT1- encoding bacteriophage VT1-Sakai [3] and the location of the corresponding prophages in the E. coli genome have been determined. The phages 933 W and VT2-Sakai integrate by site-specific recombination at the same attach- ment site, while the phage VT1-Sakai integrates at a different position in the bacterial genome. The integration of phage 933 W and VT2-Sakai disrupts the wrbA gene, which encodes the Trp repressor binding protein WrbA [1] while the phage VT1-Sakai inserts in the yehV gene [3]. Bacteriophages integrate in the bacterial genome by site- specific recombination between specific attachment sites on the bacterial (attB) and bacteriophage (attP) genomes. The integrase (Int) recognizes two distinct sequences within the attachment site. A small amino-terminal domain of the protein recognizes a nucleotide sequence motif, called the arm binding site, which is present in the attP site but not the attB site [4,5]. In addition, the Int protein contains a catalytic domain that recognizes a second motif, called the core-binding site, which is present in both attP and attB. The C-terminal region of the integrases shows pronounced differences in sequence and structure. The hallmark of the E Int family is the R-H(Y)-R-Y motif that is conserved in all members and constitutes the active site [6]. Differences in the position of the catalytic tyrosine and the surrounding secondary structure may underlie the differences in DNA cleavage [7]. The excisionase (Xis) is needed to excise the prophage from the bacterial chromosome in cooperation with the 0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0167-4781(02)00539-0 * Corresponding author. Department of Immunology, Parasitology, and Ultrastructure, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium. Tel.: +32-2-358-21-94; fax: +32-2-359-02-31. E-mail address: hdegreve@vub.ac.be (H. De Greve). 1 Present address: The Research Center of Life Sciences, Yuhuangding Hospital, Yantai 264000, PR China. www.bba-direct.com Biochimica et Biophysica Acta 1579 (2002) 196 – 202