Gene, 132 (1993) 95-99 (cl 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-I 119/93/$06.00 GENE 0728 1 95 Isolation of enterohemolysin (Ehly2)-associated sequences encoded on temperate phages of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Escherichia coli (Recombinant DNA; virulence; pathogenesis; lambda; bacteriophage) Lothar Beutin”, Uwe H. Stroeherb and Paul A. Manningb zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK “Escherichia coli Reference Laboratory, Robert-Koch-lnstitut, BGA. 1000 Berlin 6.5. Germany; and b Department of Microbiology and Immunology, University of Adelaide, Adelaide, S.A. 5001, Australia. Tel. (61-8) 228-5974 Received by M.W. Heuzenroeder: 14 January 1993; Revised/Accepted: 5 March/l9 March 1993; Received at publishers: 24 May 1993 SUMMARY We have cloned and sequenced the enterohemolysin (ehl)-associated region from a temperate bacteriophage isolated from an Escherichia coli 026:Hll strain. Phage +C3208 was isolated together with other temperate bacteriophages which transduce the enterohemolytic phenotype to non-hemolytic E. coli 026 strains. The nucleotide sequence of the 124%bp &3208 DNA insert in plasmid pE039, which mediates Ehly2 production in E. coli K-12, was determined and was found to be partially homologous to DNA of bacteriophage h but is completely unrelated to DNA sequences encoding the synthesis of Ehlyl [Stroeher et al. Gene 132 (1993), 89-941. It was shown that part of this region can be used as an Hly2-associated specific DNA probe. INTRODUCTION An enterohemolysin (Ehly) has been previously described that is epidemiologically, genetically, serologi- tally and phenotypically clearly discernible from the well- characterized Escherichia coli cr-Hly (Beutin et al., 1986; 1988; 1989). This activity is found in a number of serologi- tally different strains of E. coli which were isolated from diseased humans and from feces of healthy cattle (Beutin Correspondence to: Dr. L. Beutin, Escherichia coli Reference Laboratory, Robert-Koch-Institut, BGA, Nordufer 20, 1000 Berlin 65, Germany. Tel. (49-30) 45472484; Fax (49-30) 45472328. Abbreviations: aa, amino acid(s); Ap, ampicillin; bp, base pair(s); ehl, enterohemolysin-associated gene (DNA); Ehly, enterohemolysin or enterohemolytic; EPEC, enteropathogenic Escherichia coli; Hly, hemo- lytic or hemolysin: kb, kilobase or 1000 bp; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; PBS, phosphate-buffered saline (130 mM NaC1/7 mM K,HPO,/Z mM KHIPO, pH 7.2); PCR, polymerase chain reaction; pfu, plaque-forming unit(s); RBS, ribosome-binding site; RFLP, restriction-fragment length polymorphism(s); SDS, sodium dodecyl sulfate; [ 1, denotes plasmid- carrier state. et al., 1989; Beutin and Montenegro, 1990). In E. coli 026 strains, the expression of Ehly is directed by temperate bacteriophages. Recently, an Ehly-transducing bacterio- phage of the 026:H- strain C3888 was characterized, and the genetic determinant encoding Ehly production (Ehly-determinant) was cloned (Beutin et al., 1990). Analysis of the clone expressed in different strains and of the nt sequence revealed an ORF of approximately 30 kDa that was shown to correlate with the Ehly activity (Stroeher et al., 1993). This 30-kDa protein has been des- ignated Ehlyl. A 464-bp BamHI-EcoRV fragment that covers part of the DNA encoding the 30-kDa protein was used as an Ehlyl-specific probe in colony hybridization assays with different E. coli strains (Stroeher et al., 1993). Of 69 Ehly+ strains, only seven hybridized with this probe, indicating that most of these strains produce an Ehly which is genet- ically different from the one (Ehlyl) produced by the E. coli 026 strain C3888 (Stroeher et al., 1993). The aims of this study were to isolate, from a temperate bacteriophage, a region associated with the production of a second Ehly and to develop a specific gene probe.