For selleck inhibitor example, the OM lipoprotein Pal-mCherry MLN0128 nmr [20] localizes to mid-cell and complements a Pal deletion, and PulD-mCherry [21] allows the formation of PulD multimers in the OM. Table 1 Strains and plasmids Strains Genotype Reference LMC500 (MC4100 lysA) F, araD139, Δ (argF-lac)U169,

deoC1, flbB5301, ptsF25, rbsR, relA1, rpslL150, lysA1 [23] DH5α F, endA1, hsdR17(rk mk+), supE44, thi-1, recA1, gyrA, relA1, Δ (lacZYA-argF)U169, deoR, Ф80 lacZΔ M15 Lab collection DH5α-Z1 DH5α LacIq + TetR+ [24] Plasmids Proteins expressed Reference pGI10 pTHV037 OmpA-LEDPPAEF-mCherry This work pGV30 pTHV037 OmpA-177-(SA-1)-LEDPPAEF-mCherry This work pSAV47 pTHV037 mCherry-EFSR [25] pTHV037 pTRC99A with a weakened IPTG inducible promoter [26] Cells are grown in EZ defined rich medium [27] (see also Methods), with 0.2% glucose as carbon source. We refer to this medium as DRu (defined rich glucose) medium from now on. No adverse effects on growth rate were observed for either construct under the experimental growth and induction MM-102 ic50 conditions reported here. LMC500 (MC4100 LysA) cells expressing either construct exhibit a red fluorescent halo along the

cell’s perimeter (Figure 1A and Figure 2), as expected for fluorescence originating from the periplasm [28]. For cells grown to steady state, the fluorescence was distributed evenly along the cell perimeter, showing no preference for the cell pole, the cylindrical part or the division site. We tested if the truncate OmpA-177-(SA-1)-mCherry fusion was properly inserted in the OM using two different methods: (a) fluorescent imaging of live cells after staining the surface-exposed epitope tag, and (b) SDS-PAGE gel-shift experiments.

Figure 1 OmpA-177-(SA-1)-mCherry is properly inserted in the OM. A) Cells Dichloromethane dehalogenase grown to exponential phase in DRu medium with 0.1 mM IPTG were labeled with fluorescent streptavidin. Scale bar is 1 × 2 μm. B) mCherry-EFSR is not heat-modifiable. Sonicated cell lysate of LMC500 expressing mCherry-EFSR was resuspended in sample buffer and either; not heated (RT), heated at 37°C for 5 min, heated at 50°C for 15 min, or heated at 99°C for 10 min. Shown is an immunoblot probed with anti-DsRed antibody. The faint band present in each lane is aspecific. The unfolded (denatured) mCherry-EFSR band is indicated. Percentage of unfolded mCherry-EFSR are indicated, assuming that after heating at 99°C all protein is unfolded. C) Heat-modifiability of OmpA-177-SA-1-mCherry. Cells from the same culture used for labeling in A) were sonicated and resuspended in sample buffer. Heat treatment as in B), heating at 60°C and 70°C was for 15 min. The folded and unfolded forms of both the intact fusion and the degradation product are indicated by a preceding f- or u-, respectively. Figure 2 OmpA-mCherry is associated with the PG/OM layer. Cells expressing full-length OmpA-mCherry are plasmolyzed in hypertonic sucrose solution. Strain is LMC500.