Cells were harvested by centrifugation and resuspended in SDS sample buffer (SSB) [21] according to the following formula: resuspension volume (in μl) = 100 × A600 × vol harvested (in Proteases inhibitor ml). These concentrated cell lysates were diluted 1:100 in SSB for SDS-PAGE. Cell-free supernatants were concentrated ~10-fold by filtration using Centricon spin columns (Millipore, Billerica, MA, USA), and added to concentrated SSB for SDS-PAGE. Samples were

separated on 4-12% SDS-polyacrylamide gels and stained with silver to visualize protein bands [21]. SslE secretion experiments were repeated 2–4 times, and single representative gels are shown. To produce the images in Figure 2, the stained gels were digitally photographed

and gel images were enhanced using Adobe Photoshop software. Linear transformations (contrast and brightness adjustments) were applied to the images for clarity; such transformations were applied uniformly across any given gel image. Fusion protein localization by enzyme activity To measure secretion and surface display of SslE-enzyme fusions, cultures of WT and ΔpppA::FRT strains bearing the indicated plasmids were grown in LB at 37°C with aeration for 16–20 hours. Cells were harvested by centrifugation, and cell-free supernatants were removed; an aliquot of collected cells was removed and lysed using the PopCulture reagent from Novagen (Madison, WI, USA). Enzymatic activities associated with intact cells, lysed cells, and cell-free supernatants were then immediately measured. SslE-Cel45A 3-deazaneplanocin A manufacturer activity was measured using the CRACC assay [27], and mafosfamide SslE-Pel10A activity was measured using the pectate lyase assay described by Collmer [28]. Growth comparisons Phenotypic microarray experiments were performed

using an OmniLog reader (Biolog, Hayward, CA, USA) as per the manufacturer’s instructions using plate types PM-9 and PM-10. Cultures were grown at 37°C for 48 hours, and respiration data were analyzed using the PM software provided with the OmniLog reader. Strains used were wild-type W and Δgsp::FRT (unmarked deletion of gspC-M). To compare urea tolerances in 96-well plates, wild-type, Δgsp::FRT, and ΔpppA::FRT strains were cultured in 200 μl aliquots of LB containing 0, 0.9 M, or 1.15 M urea in 96-well plates (inoculated as 1:100 dilutions from LB overnight cultures). Plates were grown with shaking at 37°C in a Tecan M1000 plate reader (Durham, NC, USA). Growth and survival were followed by regular measurement of A595 for each culture. To compare urea tolerances in glass culture tubes, wild-type, Δgsp::FRT, and ΔsslE::FRT strains were cultured in 8 ml volumes of LB containing no urea or 1.15 M urea on a rolling wheel at 37°C. Biological duplicate cultures of each see more strain were inoculated with 1:1000 dilutions from LB overnight cultures after verification that all overnight cultures grew to equivalent A600 turbidity readings.