Genetic Manipulation in Bacteria

All oligonucleotides used are listed in Table 1. Template plasmid pWRG100 was generated using pKD3 [1] (link) as template in a PCR with primers pKD-for and pKD3-I-SceI-XbaI-rev. The resulting fragment harboring the chloramphenicol resistance gene and an I-SceI recognition site was cloned into pKD3 backbone via XbaI. The orientation of the resistance gene according to original pKD3 and its integrity was verified by restriction analysis and sequencing (not shown). Functionality of I-SceI recognition site was proven by digestion with recombinant I-SceI (Fermentas, St. Leon-Rot, Germany) (data not shown). The λ Red- and I-SceI-expressing plasmids pWRG24 and pWRG99 are derivatives of pKD46 [1] (link). The gene for I-SceI under control of a tetracycline-inducible promoter (P_tetA/tetR) was amplified by PCR from plasmid pST98-AS [14] (link) using primers NcoI-tetR-for and NcoI-I-SceI-rev and cloned in pKD46. Clones of both orientations, pWRG24 (tetR-I-SceI) and pWRG99 (I-SceI-tetR), were isolated and approved by sequencing. For complementation of strains WRG6 and WRG23 a low-copy plasmid harboring phoPQ under its natural promoter was generated. A fragment containing P_phoP-phoPQ was amplified by PCR from wild-type (WT) genomic DNA using primers XhoI-PhoPQ-for and PhoPQ-HindIII-rev. The PCR product was cloned into low-copy pWSK29 [26] (link) leading to pWRG103. All constructs were verified by restriction analysis and DNA sequencing and introduced in competent cells by electroporation (MicroPulser, Bio-Rad, Munich, Germany).