Fig 5.

RARP-1 is secreted into extracellular milieu by E. coli in a TolC-dependent manner, and R. typhi TolC restores RARP-1 secretion in tolC deficient E. coli strain. (A) Wild-type E. coli C600 and an isogenic tolC mutant harboring the indicated plasmids were grown to mid-log phase, and protein expression was induced by the addition of IPTG to a final concentration of 1 mM at 16°C. Following centrifugation of culture aliquots, proteins in the supernatants were precipitated with trichloroacetic acid. The total cell lysate equivalent to approximately 1.0 OD₆₀₀ unit and concentrated (100-fold) culture supernatant equivalent to 2 ml of culture supernatant prior to concentration were loaded on each lane and subjected to SDS-PAGE. RARP-1 was detected by Western blotting using anti-RT0218 antibodies. The recombinant RARP-1 with a C-terminal His₆ tag was purified from the total cell lysate of C600/pTrc-RT0218 using Ni-NTA agarose and used as a positive control. Lane 1, recombinant purified RARP-1; lane 2, cell lysate of C600/pTrc-RT0218; lane 3, cell lysate of C600 ΔtolC/pTrc-RT0218; lane 4, cell lysate of C600 ΔtolC/pTrc-RT0216-RT0217-RT0218; lane 5, supernatant of C600/pTrc-RT0218; lane 6, supernatant of C600 ΔtolC/pTrc-RT0218; lane 7, supernatant of C600 ΔtolC/pTrc-RT0216-RT0217-RT0218; lane 8, cell lysate of C600/pTrc-lacZ (for control plasmid); lane 9, supernatant of C600/pTrc-lacZ. Results are representative of three independent experiments. The arrow indicates the expected position of RARP-1. The molecular mass markers (lane M) are shown in kDa on the left. (B) C600/pTrc-RT0218 or C600 ΔtolC/pTrc-RT0216-RT0217-RT0218 was grown to mid-log phase and induced by the addition of IPTG for expression of a full-length RARP-1 fusion protein with His₆ tag at the C terminus. The bacterial cell lysates were prepared by collecting culture at indicated time points, and the protein was detected by Western blotting using anti-RT0218 antibodies. The molecular mass markers (M) are shown in kDa on the left. (C) Total RNA was isolated from the tolC-complemented strain after protein expression was induced overnight at 16°C. One-step RT-PCR was performed using primers designed for targeted genes (as described in the legend of Fig. 2A). Controls included the PCR analysis of the plasmid DNA isolated from the complemented strain and a no-RT reaction. The anticipated sizes of the RT0216, RT0218, and RT0216-RT0217-RT0218 amplicons were identified, indicating the expression of RT0218 and RT0216 individually and the cotranscription of all three genes of the operon.