Another genomic fragment containing sciP and adjacent ctrA was amplified using primers sciP-comF and ctrA-comR for disrupting both genes. The KIXX cartridge replaced a 644-bp SmaI/BamHI fragment, deleting the last 215 bp of sciP and the first 331 bp of the 711 bp ctrA. Plasmids containing disrupted versions of the genes were conjugated to R. capsulatus from E. coli C600 (pDPT51) (Taylor et al., 1983). Mutant strains were generated by GTA transfer of the disrupted versions of the genes into the chromosome of SB1003 (Scolnik & Haselkorn, 1984). PCR

with the original amplification primers was Linsitinib used to confirm the resulting kanamycin-resistant strains contained only the disrupted genes. Plasmid recombineering (Noll et al., 2009) was used for the generation of the cckA deletion construct. Primers cckA-p1 and cckA-p2 (Table 1)

were used to amplify a 4351-bp region encoding cckA (rcc01749) plus ~1 kb of flanking Metformin in vitro sequence on each side. Gel-purified PCR fragments were recombined into pUC19 (Vieira & Messing, 1982), and parental plasmids were selectively linearized by SalI treatment. Primers cckA-p3 and cckA-p4 (Table 1) were designed to PCR-amplify kanamycin resistance cassette A002 (Gene Bridges, Germany) with 50-bp tails homologous to cckA bases 53–103 and 2202–2252, respectively. λ-Red recombination resulted in the replacement of ~91% of plasmid-encoded cckA with the kanamycin resistance marker, yielding pUCΔcckA. The resulting plasmid was used to generate the cckA mutant strain as described above for the chpT, sciP and the ctrA/sciP double mutants.

Trans complementation of wild-type genes under control of their native upstream sequences was performed with the low copy number broad-host-range plasmid, pRK767 (Gill & Warren, 1988). The complementing fragments were amplified from the genome with appropriate primers (Table 1). Site-directed mutagenesis was performed with the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) to create pRK767-borne mutant ctrA genes with their native promoter region encoding a CtrA phosphomimetic protein, CtrAD51E (Domian et al., 1997), and a CtrA protein that is unable to be phosphorylated, CtrAD51A (Ryan et al., 2002), using primers D51E-F/D51E-R Amrubicin and D51A-F/D51A-R, respectively (Table 1). The mutagenesis created single bp substitutions that resulted in a glutamate (D51E) or alanine (D51A) in place of the conserved aspartate (D51) phosphorylation site; the presence of the mutations was confirmed by sequencing. These plasmids and the empty pRK767 control were transferred to R. capsulatus via conjugation using E. coli S17-1 (Simon et al., 1983). RcGTA packages random fragments of the R. capsulatus genome and transfers these to recipient cells. A gene transfer bioassay was used to measure production and release of RcGTA particles.