Figure 2. AzF is site-specifically incorporated into SifA-E52TAG in Salmonella.

(A) Left: Coomassie stained SDS-PAGE; right: SDS-PAGE fluorescence imaging confirms selective labeling of SifA-E52AzF with BDP-DBCO in the cell lysate. (B) Expression of SifA-E52AzF in Salmonella analyzed by fluorescence microscopy in the presence (top) or absence (bottom) of 1mM AzF. Salmonella cells expressing SifA-E52AzF in the presence or absence of AzF were incubated with BDP-DBCO, and imaged for BDP fluorescence (green). SifA fluorescence (green) is only observed in the presence of AzF. (C) Site-specific incorporation of TCO*Lys into SsaP-Y65TCO in Salmonella left: Coomassie stained SDS-PAGE; right: SDS-PAGE fluorescence imaging confirms selective labeling of SsaPY65TCO with BDP-Tz in the secreted fraction collected after TCA precipitation. (D) Expression of SsaP-Y65TCO in Salmonella analyzed by fluorescence microscopy in the presence (top) or absence (bottom) of 1mM TCO*Lys. Salmonella cells expressing SsaP-Y65TCO in the presence or absence of TCO*Lys were incubated with SiR-Tz, fixed at 8 hr post acid induction and imaged for SiR fluorescence (magenta). Polarly localized SsaP fluorescence (magenta) is only observed in the presence of TCO*Lys. A higher magnification merged imaged is shown in the inset. See Figure 2—figure supplement 5 for statistical analysis for the fraction of polarly localized SsaP. Images were acquired using confocal microscopy. The data are representative of at least three independent experiments. BF = bright field. Scale bar, 2 µm (B,D), 1 µm (inset).

Figure 2—figure supplement 1. SsaP is required for SPI-2 secretion.

In the wild-type background, the secreted translocon protein SseB appears in both whole cell and secreted fractions (left panel, lane 1). In the ssaP null strain, SseB is produced, but not secreted. SseB secretion is restored by providing ssaP in trans at the ʎ attachment site. A fusion protein with PAmCherry at the N-terminus of SsaP does not support SseB secretion, but a C-terminal PAmCherry fusion restores secretion. GroEL was used as a loading control. The C-terminal SsaP-PAmCherry fusion appears to be cleaved as observed by western blot (right panel).

Figure 2—figure supplement 2. Selection of ncAA incorporation sites.

Residue conservation scores calculated from multiple sequence alignments were mapped onto the crystal structure of SifA (PDB ID: 3CXB) using the ConSurf web server (http://consurf.tau.ac.il/). The visual color key indicates the residue conservation score (cyan: low conservation, magenta: high conservation). The ncAA incorporation sites are highlighted in red rectangles. Protein structures were visualized in Chimera (left) and NGL 3D Viewer (right).

Figure 2—figure supplement 3. Western blot of SseB (translocon) and SsaP-HA secretion.

Whole cell and secreted fractions were harvested after growth in SPI-2-inducing conditions. The SseB translocon was secreted in the wild-type strain (far left), but not in the ssaP null strain. Complementation of ssaP restored SseB secretion. HA tags were inserted at various locations in SsaP, and the effect on secretion was monitored. HA tags at amino acid position 20, 50, and 110 (red) supported SseB secretion, whereas HA tags at position 86 and 124 did not. In the lower panels, the HA antibody identifies a diffuse band of SsaP protein in the secreted fraction, similar to the fluorescently labeled SsaP (Figure 2). Wild-type SsaP was not detected, because it lacked the HA tag. GroEL was used as a loading control.

Figure 2—figure supplement 4. AzF and subsequent Alexa555 sDIBO Alkyne incorporation is highly specific.

Cells were transformed with pEVOL and psifA52TAG-GFP or psifA-WT-GFP were grown in the presence of 1 mM AzF and 0.2% L-arabinose in MgM pH 5.6, 37°C. Cells were harvested at OD₆₀₀ = 0.4. After extensive washout of excess AzF, cells were incubated with 1 μM Alexa555-sDIBO at 37°C for 60 min. After another extensive washout of excess dye, cells were imaged on agarose pads at ambient temperature using a 488 nm (GFP signal) and 561 nm (Alexa555) excitation laser. First row, left panel shows the expression of wild-type SifA-GFP in the presence of AzF. It is not labeled by Alexa555-sDIBO (bottom panel). The middle panel shows the absence of SifA-E52AzF-GFP-containing bacteria in the absence of ncAA (top), whereas the same strain expressed SifA-E52AzF-GFP in the presence of 1 mM AzF (right). Fluorescent labeling of Salmonella expressing SifA-E52AzF-GFP with Alexa555-sDIBO revealed red fluorescence, which also corresponds to the GFP signal (compare top and bottom rows, right panel). The red fluorescence is absent in the absence of AzF (bottom row middle panel). No red fluorescence was observed in Salmonella cells expressing wild-type SifA-GFP in the presence of AzF (bottom row left panel), demonstrating the extent of SifA-specific labeling using GCE. Results presented in each panel were obtained in at least three independent experiments. Scale bar, 2 µm.

Figure 2—figure supplement 5. Average fraction of cells that display polarly localized SsaP (n_total = 112 cells).

Error bars are standard deviation across 18 ROIs pooled from three independent experiments.