Figure 6
MobC influence on efficiency of self-transmission of plasmids with RA3 conjugative module. A. DNA binding of MobC in vitro. Plasmid DNAs of pJSB2.43 (61 nt insert with OM) and pJSB2.44 (45 nt insert without OM) were digested by PvuII and incubated with different amounts of His6-MobC in 20 μl of binding buffer at 37°C for 15 minutes (0-20 pmoles). The complexes were separated on 1% agarose gels run in 1xTBE and visualized by ethidium bromide staining. Fragment 2364 nt corresponds to “unspecific” vector DNA, small fragments in both plasmid DNAs contain oriT RA3 with OM (383 nt) or without MobC binding site (367 nt). B. Schematic presentation of tested vectors carrying the RA3 conjugative module with mobCp substituted by lacI q tacp (no OM). Dark grey regions correspond to the contiguous TraRA3 region and light grey parts indicate korCp-korC fragment from RA3 maintenance module. Black regions show mobC-nic or nic genes under tac promoter control. The 45 nt oriT RA3 is indicated by black rectangular. Restriction sites used during construction of pJSB2.57 and pJSB2.58 are listed. Numbers in brackets correspond to RA3 coordinates. C. The effect of MobC presence on the conjugation frequency. The strains: DH5α(pUC18), DH5α(pJSB2.57), DH5α(pJSB2.58)(pBBR1MCS-1), DH5α(pJSB2.58)(pJSB4.1) and DH5α(pJSB2.58)(pJSB4.2) served as donors in conjugation with the recipient strain DH5α RifR. Conjugation frequency is indicated on the semilogarithmic scale as the number of transconjugants per total number of donor cells. Mean values with standard deviation of at least three experiments are shown.