4 702 hlyA (3865-3883) (4592-4613) BIRB 796 chemical structure FM180012 113f 113r CTTGGTGGCGATGTTAAGG GACTCTTTTTCAAACCAGTTCC 53.5 749 hlyD (8297-8319) & IS911 (8925-8946)

FM180012 99f 99r GCAGAATGCCATCATTAAAGTG CCATGTAGCTCAAGTATCTGAC 53.8 650 PAI I (536) (44506-44524) &hlyC (45278-45299) AJ488511 81f 81r CCTGTGACACTTCTCTTGC CCCAAGAACCTCTAATGGATTG 52.3 773a PAI II (536) (31974-31995) &hlyC (32650-32668) AJ494981 72f 72r CCCAACTACAATATGCAACAGG CGCCAATAGAGTTGCCTTC 51.9 695 a) PCR products of different lengths were obtained with these primers depending on the DNA template (see Table 1) Figure 2 Map of the α- hly region of plasmid pEO5 (FM180012). The positions of PCR-primers used for investigation of strains with plasmid and chromosomally inherited α-hly genes are indicated as leaders carrying the primer designations CUDC-907 (Table 2). Regulatory sequences inside the hlyR gene (A, B and OPS) are shown as filled ballons. “”phly152″” is a stretch of non-coding DNA showing strong homology to corresponding regions in the α-hly plasmid pHly152.

Primers 1f/r are specific for the upstream hlyC region in pEO5 and yielded a PCR product of 678 bp (Fig. 2). PCR products of the same size were obtained with all strains carrying α-hly plasmids, except 84/S (pEO14); restriction enzyme analysis revealed all the fragments had a similar HinfI profile (data not shown). Primers 1f/r gave no products using E. coli strains carrying chromosomally encoded α-hly as template with the exception of the E. cloacae check details strain KK6-16 which yielded a PCR product; DNA sequencing revealed a 778 bp fragment [GenBank FM210352, position 72-849] (Table 1). Primers 32f/r spanning the region between hlyR and the “”phly152″” segment amplify a 671 bp product in pEO5 [GenBank FM180012, position 597-1267] (Fig. 2). A PCR product of Pregnenolone the same size was obtained with pEO5

and derivative plasmids as well as with plasmids pEO9 [GenBank FM210248 position 427-1097], pEO13 and pEO860 (Table 1, Fig. 3). Primers 32f/r yielded PCR products of 2007 bp with pEO11, [GenBank FM210249, position 392-2398), pHly152 and pEO12, and 2784 bp PCR products with pEO853 [GenBank FM10347 position 399-3182], pEO855 and pEO857 (Table 1). All amplicons of a given size (671 bp, 2007 bp and 2784 bp), yielded a similar HinfI restriction pattern (data not shown). Strains with chromosomally encoded α-hemolysin gave no products in the 32f/r PCR, as well as strain 84/2 S carrying plasmid pEO14 (Table 1). Figure 3 Map of the hlyR – hlyC region of representative plasmids of groups 1, 2 and 3. Genetic map of the corresponding regions from hlyR to hlyC of α-hly determinants from plasmids representing groups 1-3. A) pEO9, (strain 84-2195) B) pEO11, (84-3208); and C) pEO853 (CB853). The positions of PCR-primers used for identification and nucleotide sequencing are indicated as leaders carrying the primer designations (Table 2). Regulatory sequences inside the hlyR gene (A, B and OPS) are shown as filled ballons.