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. 2004 Jan 8;101(3):841–846. doi: 10.1073/pnas.0304916101

Fig. 1.

Fig. 1.

Interbacterial protein translocation by the Dot/Icm system in the absence of DNA transfer. (A) Assay for interbacterial protein transfer. Translocation of Cre hybrid protein from a donor bacterial strain is measured by removal of a floxed transcriptional terminator located between the trc promoter and the npt II (kanR) gene on plasmid pZL184 harbored by a recipient bacterial strain. Bacteria harboring the intact reporter are unable to grow on media containing kanamycin and sucrose. The translocation of Cre hybrid protein into the recipient strain leads to the excision, through recombination at the loxP sites, of the DNA fragment that confers sucrose sensitivity (sacB) and reconstitution of a functional loxP-npt II translational fusion. S.D., Shine–Dalgarno sequence; npt II, neomycin phosphotransferase; red diamond, transcriptional terminator. Arrows indicate the trc promoter and loxP sites. (B) Interbacterial transfer of a fusion derived from the RSF1010 mobA gene. The mobA gene was fused to cre, and RP4-dependent protein translocation into a recipient E. coli strain was measured by using E. coli S17–1 (15) as the donor selecting kanamycin resistance and screening for gentamicin sensitivity. Black bars, S17–1 Trb+ donor; stippled bars, E. coli DH5α Trb– donor. (C). Plasmids harboring Cre fusions cannot be transferred to recipient cells. Plasmids expressing the designated proteins were harbored in either E. coli S17–1 (Trb+) or L. pneumophila Lp02 (Dot/Icm+; ref. 11), and the efficiency of plasmid transfer was measured by using either recipient E. coli or L. pneumophila strains, respectively. As a positive control, the identical plasmids having an intact oriT and mob system were used to demonstrate transfer proficiency of donor strains. Black bars, donor strain E. coli S17–1 (Trb+); gray bars, donor strain L. pneumophila Lp02 (Dot/Icm+; ref. 11). (D) Transfer of translocated Dot/Icm substrates between bacterial cells. Protein transfer was performed as described in Materials and Methods, by using either Lp02 (Dot/Icm+) or Lp03(dotA–) expressing the designated protein fusions as the donor strains. Gray bars, donor strain L. pneumophila Lp02 (Dot/Icm+); stippled bars, donor strain L. pneumophila Lp03 (dotA–).