Figure 5.

(A) Tla3(PA0259) is a cytoplasmic protein of P. aeruginosa. Cells of P. aeruginosa PAO1 tla3_V5 were subjected to fractionation and immunoblotting using antibodies directed against the V5 tag, XcpY and DsbA. XcpY and DsbA were used as membrane and periplasmic controls, respectively. T, whole cell; C, cytoplasm; Mb, total membrane; P, periplasm. (B) The annotated ATG drives the initiation of translation of tla3 in P. aeruginosa. Immunodetection of Tla3_V5 with anti-V5 antibodies produced in a WT background or in strains in which one of the four predicted ATG in tla3 have been substituted in ATA. The number followed by a star indicates which ATG from Supplementary Figure S1 ATG₄ corresponds to the annotated ATG. (C) Tla3 is not required for Hcp2b secretion. Immunodetection of Hcp2b_6His with anti-His antibodies produced in a WT background (line 1) or in strains deleted for rsmA (lines 2–4) and clpV2 (line 3) or tla3 (line 4). The strains were grown at 25°C for 24 h and total bacteria were separated from extracellular medium. Anti-EF-Tu is used as a lysis control. The extracellular medium proteins were also stained with Coomassie-blue. (D) Tla3 does not interact with Tle4. Copurification assay on StrepTactin column of Tla3^c_STREP with Tle4_His produced in E. coli BL21(DE3)pLysS from pBB27 and pVT3 respectively. Legend is as in Figure 4. (E) Tla3 is not secreted. Immunodetection of Tla3_V5 with anti-HV5 antibodies produced in a WT background (line 1) or in strain deleted for rsmA (line 2). The strains were grown at 25°C for 24 h and total bacteria were separated from extracellular medium. Anti-EF-Tu is used as a lysis control. The extracellular medium proteins were also stained with Coomassie-blue. (A‑E) The position of the proteins and the molecular mass markers (in kDa) are indicated.