Journal: PLoS Biology

Article Title: Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle

doi: 10.1371/journal.pbio.3000874

Figure Lengend Snippet: (A) In vivo expression of plasmid-encoded YohP, AzuC, YshB, and YkgR. Expression was induced by 0.2% arabinose (“Ara”) when indicated and whole cells were TCA precipitated, before SDS-PAGE and western blotting using α-His antibodies. The predicted amino acid sequence is shown on the right and the putative transmembrane domains are indicated in bold. Positively charged residue are shown in red. (B) YohP-GFP, SecY-YFP, and the cytosolic control protein YchF-GFP were in vivo expressed and exponentially grown cells were analyzed with a DeltaVision Ultra High Resolution Widefield Microscope (GE Healthcare, Munich, Germany) at 100× magnification. Recording, using camera sCMOS pro edge (PCO, Kelheim, Germany), was performed using a 3-μm Z-scan with 0.1-μm sectioning, and the different scans of both the fluorescence channel and the merged fluorescence/bright-field picture is shown. (C) WT E . coli cells and cells expressing YohP His from the IPTG-inducible pET19b-YohP His were analyzed by transmission electron microscopy. (D) E . coli cells expressing YohP His were induced with 1 mM IPTG when indicated and subsequently fractionated by differential centrifugation. S30/P30 refer to the supernatant/pellet after the first centrifugation (30,000 g ) following cell breakage. The S30 fraction was then further centrifuged (150,000 g ), and the observed pellet (P150, containing crude membranes) was subjected to sucrose gradient centrifugation, separating the INV and the OM. Of each fraction, an aliquot (40 μg protein) was separated by SDS-PAGE and, after western transfer, decorated with α-His antibodies (YohP His ), α-SecY, α-SecG, and α-YchF antibodies. A representative image of two independent biological replicates is shown. GFP, green fluorescent protein; INV, inner membrane vesicle; IPTG, isopropyl 1-thio-β-D-galactopyranoside; OM, outer membrane fraction; TCA, trichloroacetic acid; WT, wild type; YFP, yellow fluorescent protein; YohP His , His-tagged YohP.

Article Snippet: Imaging was performed with a DeltaVision Ultra High Resolution Widefield Microscope (GE Healthcare, Munich, Germany) at 100× magnification with an Olympus UPlanSapo objective (Olympus, Hamburg Germany; numerical aperture 1.35) and immersion oil with a refractive index of 1.5120.

Techniques: In Vivo, Expressing, Plasmid Preparation, SDS Page, Western Blot, Sequencing, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy, Centrifugation, Gradient Centrifugation

Journal: PLoS Biology

Article Title: Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle

doi: 10.1371/journal.pbio.3000874

Figure Lengend Snippet: (A) E . coli wild-type cells carrying pBad24-MS2-GFP were induced with 0.1 mM arabinose and 5 μL culture solution were placed on a sterile glass-bottom dish (35-mm dish with 20-mm bottom well, glass thickness 0.16–0.19 mm). Imaging was performed with the DeltaVision Ultra microscope (exposure 0.2 seconds for GFP at 35% laser intensity and 0.075 seconds for bright field at 5% laser intensity), and 3-μm Z-scans were recorded with an interval of 1 μm. The displayed images were taken at the focal point. The image was developed with the ImageJ Fiji software. (B) As in (A) but cells were coexpressing the pSC-bglB-MS2.6x plasmid or the pSC-yohP-MS2.6x plasmid, encoding the bglB or yohP mRNA, respectively, each with a deleted ribosome binding site and a hexa-repeat MS2 stem-loop recognition sequence at the 3′ UTR. Expression of the respective mRNAs was induced at an OD 600 = 0.5 with 4 mM IPTG, and MS2-GFP was induced 40 minutes after IPTG addition with 0.1 mM arabinose. Samples were analyzed as above and Nile red was used to stain the membrane. Nile red staining was monitored with an exposure time of 0.2 seconds at a laser intensity of 35%. GFP, green fluorescent protein; IPTG, isopropyl 1-thio-β-D-galactopyranoside.

Article Snippet: Imaging was performed with a DeltaVision Ultra High Resolution Widefield Microscope (GE Healthcare, Munich, Germany) at 100× magnification with an Olympus UPlanSapo objective (Olympus, Hamburg Germany; numerical aperture 1.35) and immersion oil with a refractive index of 1.5120.

Techniques: Imaging, Microscopy, Software, Plasmid Preparation, Binding Assay, Sequencing, Expressing, Staining