Expression of functional Tat substrates causes TatC to cluster at the cell poles.

<p>(<b>A</b>) <i>E. coli</i> BL21(DE3) wild type cells (wt) expressing TatC-mCherry from a pBAD33 vector (pPR3). Expression was induced by the addition of 0.1% arabinose for 2 h. Overall weak fluorescent signal that in some cells is concentrated at the cell periphery (arrow). (<b>B</b>) Co-expression of TatC-mCherry from a pBAD33 vector (pPR3) as in (A) and of the Tat substrate TorA-MalE335 from a pET22 vector (pPJ1) at non-induced levels. The additionally expressed Tat substrate causes a pronounced clustering of TatC-mCherry at the cell poles. (<b>C</b>) Co-expression of TatC-mCherry from a pBAD33 vector (pPR3) as in (A) and of the natural Tat substrate SufI from a tightly controlled pASK-IBA33plus vector (pPJ9) by the subsequent addition of 50 ng/ml anhydro-tetracycline. Samples were taken at the indicated times and inspected under the microscope. Appearance of polar clusters starts after 30 min with a maximum reached after 50 min. No clustering of TatC-mCherry occurs without induction of SufI. (<b>D</b>) as in (B) expressing the non-functional KK-version of TorA-MalE335 from plasmid pPJ2. This does not lead to re-localization and clustering of TatC-mCherry at the poles. (<b>E</b>) as in (B) except that after inducing synthesis of TatC-mCherry and TorA-MalE335, 100 µM CCCP was added to the growth medium 30 min before cells were microscoped. Dissipation of the PMF does not interfere with the TorA-MalE335-dependent agglomeration of TatC-mCherry at the cell poles. (<b>F</b>) as in (B) except that TatC-mCherry and TorA-MalE335 were expressed in BL21(DE3) Δ<i>tat</i> cells (Δ<i>tat</i>). The TorA-MalE335-mediated clustering of TatC-mCherry at the cell poles is also independent of TatA and TatB. (<b>G</b>) Equal expression levels of TatC-mCherry in the cells depicted in the indicated panels. After 2 h of induction, whole cell proteins were precipitated with trichloroacetic acid and equivalent amounts were subjected to SDS-PAGE. Immunoblots were decorated with antibodies against TatC.