In addition, transconjugants were tested for CI formation (PaiII_1rev/PaiII_53fw) and for site-specific integration of PAI II536 into the tRNA gene leuX (M803b/M805c). The latter two primer pairs Y-27632 research buy also allowed the determination of the orientation of the integrated PAI (Figure 2). Remobilization experiments were carried out with two PAI II536-positive clones of E. coli K-12 as donors that derived from the mobilisation experiments and
a derivative of the wild type UPEC strain 536 as recipient. Donor and recipient strains were mixed in a 3 : 1 ratio and incubated at 20°C and 37°C, respectively. Experiments were divided into two sets according to the state of the mobilised PAI. In the first set, the donor strain SY327-77
harbored PAI II536 in the circular form. In the second set, clone SY327-23, harboring the chromosomally integrated PAI II536, served as donor strain. In both ML323 clinical trial cases, strain 536-21, a non-hemolytic derivative of strain 536, which lacks the two islands encoding functional α-hemolysin determinants (PAI I536 and PAI II536) , served as the recipient. In the remobilisation experiments, the same PCR-based verification process as described above was carried out with ATM/ATR inhibitor drugs exconjugants that grew on the Cm-lactose-M9 minimal agar plates. In addition, pulsed-field gel electrophoresis (PFGE) analysis of the randomly picked transconjugants was carried out. Genomic DNA for PFGE analysis was prepared and cleaved with NotI or SfiI as described before . Gels were run for 21-24 h with pulse times of 0.5-50 s. Phenotypic characterisation of transconjugants PAI II536 comprises a α-hemolysin gene cluster. This determinant was used as a phenotypic marker in this study to verify the presence of PAI II536 in transconjugants after the mobilisation and remobilisation experiments. Therefore, transconjugants were screened post-experimentally on blood agar plates to analyse the hemolytic activity. UPEC strain 536 served as a positive control, while strains SY327 and 536-21 served as negative controls. Statistical analysis Statistical analysis of the conjugation rate was performed
by the Mann-Whitney U test. The ratio/distribution of integrated, cointegrated and partial transconjugant clones at 20°C and 37°C was compared by the chi-square test. The difference was considered significant if p < 0.05. Dynein Acknowledgements and Funding This work was carried out within the European Virtual Institute for Functional Genomics of Bacterial Pathogens (CEE LSHB-CT-2005-512061) and the ERA-NET PathoGenoMics I consortium “”Deciphering the intersection of commensal and extraintestinal pathogenic E. coli”" (Federal Ministry of Education and Research (BMBF) grant no. 0313937A) and the Hungarian Research Foundation (OTKA 62092 and 78915). UD was also supported by the German Research Foundation (DO 789/4-1). The excellent technical assistance of K. Lotzl (Pécs) and B. Plaschke (Würzburg) is appreciated.