coli (UPEC) strains  and with enterotoxigenic (ETEC), shigatoxigenic (STEC) and enteropathogenic E. coli (EPEC) strains that cause diarrhea and edema disease in animals [9–12]. In UPEC the α-hly genes are found on
large chromosomal pathogenicity islands (PAI) [13, 14]. The UPEC O4 (J96) and O6 (536) strains carry each two α-hly operons located on different PAIs [15, 16], which contain divers junctions and adjacent sequences. This suggests that these loci have evolved independently [16, 17]. Genetic analysis of chromosomal α-hly operons revealed differences in 5′ flanking Vorinostat sequences and toxin expression [18–20]. Plasmid-encoded α-hly genes were found associated with EPEC O26 strains , as well as with ETEC and Shiga toxin 2e (Stx2e) producing STEC strains [9, 10, 22]. α-hly plasmids of E. coli were found to differ widely in size, incompatibility groups and conjugational transfer ability [10, 20, 21, 23]. So far, only two plasmid α-hly operons were completely sequenced. The first is located on the 48 kb non-conjugative plasmid pHly152 from a murine E. coli Dibutyryl-cAMP strain . The other is located on the 157 kb conjugative plasmid pEO5 of a human EPEC O26 strain . Interestingly, despite the differences between pHly152
and pEO5, the DNA sequence of their α-hly operons are 99.2% similar while the sequence of the upstream regulatory hlyR region is 98.8% similar . Importantly, PX-478 the plasmid-inherited Megestrol Acetate α-hly are less similar (96.0-96.4%) to the chromosomally inherited
α-hlyCABD located on PAI I [GenBank AJ488511] and PAI II [GenBank AJ494981] of the E. coli strain 536 [18, 21]. Moreover, chromosomally and plasmid-inherited α-hly operons also differ also for their 5′ regulatory hlyR region. These findings suggest that the plasmid and chromosomal α-hly operons have evolved in parallel. Studies on hemolysins of other bacterial species revealed similarities between the E. coli α-hemolysin genes and the Enterobacter, Proteus, Morganella and Mannheimia operons [25, 26]. Codon usages base composition studies suggested that the α-hlyCABD genes of E. coli were originated from Proteus, Morganella or Mannheimia species [25, 27]. Transposon-like structures found in the neighborhood of plasmid pHly152 and pEO5 encoded α-hly operons suggest that these were acquired by horizontal gene transfer [20, 21]. The fact that the α-hlyCABD genes and their adjacent regions on pHly152 and pEO5 were highly similar to each other prompted us to investigate the genetic relationship between plasmid and chromosomal inherited α-hly operons in more strains of E. coli and in Enterobacter cloacae. Our results indicate that plasmid α-hly operons are highly similar regardless of differences in the plasmid backbone sequences, bacterial host and their source, suggesting that they have evolved from a common origin. Results Characterization of α-hly plasmids in E.