The relevant characteristics of strains with chromosomally located α-hemolysin determinants are listed elsewhere [10, 18, 19]. The α-hemolytic E. cloacae strain KK6-16 as well as the canine and porcine ETEC and STEC strains carrying α-hly XL184 solubility dmso plasmids were described previously [10, 26, 29, 42]. The EHEC-hemolysin plasmid pO157 carrying strain TPE1313 was used as negative control is described elsewhere [21]. Mating of bacteria with E. coli K-12 recipient strains and isolation of α-hemolytic transconjugants was selleck inhibitor performed as described by Burgos et al. 2009 [21]. Phenotypes corresponding to E. coli α-hemolysin were

analyzed on washed sheep blood agar [43]. Isolation of DNA, RNA and cDNA Total DNA of bacteria was isolated as described [29]. Purified plasmid DNA of bacteria that was used for restriction digestion, DNA-hybridization, PCR and nucleotide sequencing was isolated with the large construct kit following the instructions of the producer (Qiagen, Hilden, Germany). Analysis of total plasmid profiles of E. coli strains was performed as described previously [44]. Total RNA was isolated from 20 ml of exponentially growing aerated cultures (3-5 × 108 bacteria/ml) of bacteria in L-Broth with the RNeasy minikit (Qiagen). Isolation of RNA and preparation of cDNA was performed as described previously [29]. DNA hybridization Southern blot hybridization of plasmid DNA and labeling

of gene probes with Digoxigenin-11-dUTP Selleck RG7420 was performed as described [21]. Dig-labeled molecular markers (Dig Roche) were used for size determination of hybridizing DNA fragments. For identification of α-hly plasmids in Southern blotted gels a 666 bp

PCR product of the α-hlyA gene generated with primers 10f/r (Table 2) was used as internal DNA probe for detection of α-hly specific sequences [21]. Plasmids pHly152, pO157 and pEO5 served as reference plasmids for size determination of α-hly plasmids [21] (Fig. 1). Nucleotide sequencing of α-hemolysin and associated sequences Nucleotide sequence analysis of the α-hly determinants and adjacent sequences was performed as described [21]. PCR products were purified and used Janus kinase (JAK) for sequencing applying the dye terminator chemistry (PE Applied Biosystems, Darmstadt, Germany) and separated on an automated DNA sequencer (ABI PRISM® 3100 Genetic Analyzer, Applied Biosystems, Foster City, CA). The sequences were analyzed using the Lasergene software (DNASTAR, Madison,WI) and Accelrys Gene v2.5 software. Development of specific PCRs for plasmid- and chromosomally inherited α-hly determinants and their associated sequences Primer pairs specific for α-hly-plasmid specific sequences hlyR (primers 44f/r), the region between hlyR and hlyC (primers 1f/r, 32f/r), hlyA (111f/r and 113f/r) and hlyD and downstream (99f/r) (Table 2) were developed with Accelrys software using the pEO5 sequence [GenBank FM180012].