Figure 4
Modifications of MobC operator. A. Scheme of PCR site-directed modifications in mobCp region. Sequences of modified motifs (IR3 and IR4) are shown with the modifications underlined and in bold. Mutant alleles (mutVI-IX) were PCR-amplified and analyzed in EMSA (panel B), cloned into promoter-probe vector pPT01 upstream of xylE cassette (panel C and D). B. DNA binding activity of MobC in vitro. Two picomoles of 417-bp DNA fragments amplified by PCR on templates shown in panel A were incubated with 0 to 20 picomoles of His-tagged MobC in 20 μl of binding buffer at 37°C for 15 minutes. The complexes were separated on 1.2% agarose gels run in 1xTBE and visualized by ethidium bromide staining. The bottom panel refers to EMSA of MobC with unspecific DNA fragment, PCR-amplified klcAp of RA3 (coordinates 2336-2704 nt). C. MobC regulation of mobCp derivatives in vivo. DH5α strains carrying pJSB7.9 (wt 417 bp fragment) or its mutant derivatives (labeled according to panel A) were transformed with empty vector pGBT30 (light grey bars) or pJSB5.1 tacp-mobC (dark grey bars). Diagram presents XylE activities in extracts of the double transformants relative to the XylE activity detected in extract of DH5α(pJSB7.9)(pGBT30) strain. Mean values with standard deviation of at least three assays are shown. D. DH5α (RA3) strain was transformed with pPT01 derivatives carrying substitution mutant variants of mobCp region. Double transformants were used as donors in conjugation with DH5α RifR strain as the recipient. The frequency of mobilization is indicated on a semi-logarithmic scale as the number of transconjugants/donor cell, where vector corresponds to empty pPT01, wt to pJSB7.9 (mobCp-xylE), and roman numerals to the mutants presented in panel A. Mean values with standard deviation of at least three experiments are shown.