, 2009). DNA was immediately extracted from 1 mL of this cell suspension and the gfp gene was quantified using the primer pair gfp_F2/R2, and pRE25* using the primer set pRE25*_F2/R2 and the TaqMan probe pRE25*_TMP. The ermB gene on pRE25 plasmid was marked to allow quantification of the gene and to monitor transmission routes in conjugation experiments in complex genetic backgrounds as for example the GI-tract. Therefore, L. lactis BuRE25 (Table 1), harboring pRE25 in its chromosome, was transformed with the integration vector pMH401 (Fig. 1), and a double-cross-over event resulted in pRE25*, a pRE25-derivative harboring tet(M) flanked by two selleck screening library random sequences. Next, pRE25*
was transferred to E. faecalis CG110/gfp (Scott et al., 2000; Table 1) via filter mating, resulting in strain E. faecalis CG110/gfp/pRE25*, a new tool for monitoring and quantification gene transfer in complex microbial environments. Even in the postgenomic era, classical manipulation of large DNA molecules is still inefficient due to technical limitations in purification, size separation, and handling, (Gibson et al., 2010.), and initial attempts to manipulate the 50-kb plasmid pRE25 directly were not successful. Lactococcus lactis BuRE25 harbors pRE25 in its chromosome (Perreten, 1995) and this allowed a relatively easy manipulation of the plasmid via homologous recombination.
Moreover, L. lactis BuRE25 is tetracycline sensitive, thus providing use of the additional selection marker tet(M). Natural Product Library ic50 The two markers in strain E. faecalis CG110/gfp/pRE25*, gfp and the random sequences on pRE25*, are usually not present in the human intestine,
allowing one to distinguish a donor strain from transconjugants in complex background flora by molecular methods such as quantitative PCR. Strain E. faecalis CG110/gfp/pRE25* harbors a number of ABR genes, and we initially analyzed the presence and function of these genes to characterize the strain. Acyl CoA dehydrogenase Hybridization using a microarray harboring probes for 90 different ABR genes confirmed the presence of resistance genes against tetracycline, erythromycin, streptothricin, kanamycin, and streptomycin, whereas the presence of cat in strain CG110/gfp/pRE25* was confirmed by PCR (data not shown). Microdilution test showed phenotypic resistance of strain CG110/gfp/pRE25* to chloramphenicol, erythromycin, gentamicin, kanamycin, rifampicin, streptomycin, and tetracycline, with a lower MIC for chloramphenicol and streptomycin compared with E. faecalis RE25 (Table 3). Phenotypic resistance of CG110/gfp/pRE25* to rifampicin is due to the chromosomally encoded resistance of the host strain CG110 (Jacob & Hobbs, 1974). Tetracycline resistance is encoded on the chromosome and on the plasmid, whereas the other resistance genes are encoded only on pRE25*.