Adaptation of the MultiSite Gateway Pro 3-fragment recombination for use in bacteria.

<p>Two Entry clones containing each a gene of interest (GOI) recombine (crossed grey lines) together with a third Entry clone and one Destination vector harboring each a reporter gene (R1/2) to create an Expression clone. The Expression clone contains three DNA elements donated from the three Entry clones and enables expression of the GOI1-R1 and GOI2-R2 fusion genes from tetracycline-inducible promoters (P_<i>tetA</i>). Transcription of fusion genes is stopped by a termination sequence derived from that of the <i>rrnB</i> operon (Ω).</p

A Gateway-Based System for Fast Evaluation of Protein-Protein Interactions in Bacteria

<div><p>Protein-protein interactions are important layers of regulation in all kingdoms of life. Identification and characterization of these interactions is one challenging task of the post-genomic era and crucial for understanding of molecular processes within a cell. Several methods have been successfully employed during the past decades to identify protein-protein interactions in bacteria, but most of them include tedious and time-consuming manipulations of DNA. In contrast, the MultiSite Gateway system is a fast tool for transfer of multiple DNA fragments between plasmids enabling simultaneous and site directed cloning of up to four fragments into one construct. Here we developed a new set of Gateway vectors including custom made entry vectors and modular Destination vectors for studying protein-protein interactions via Fluorescence Resonance Energy Transfer (FRET), Bacterial two Hybrid (B2H) and split <i>Gaussia</i> luciferase (Gluc), as well as for fusions with SNAP-tag and HaloTag for dual-color super-resolution microscopy. As proof of principle, we characterized the interaction between the <i>Salmonella</i> effector SipA and its chaperone InvB via split Gluc and B2H approach. The suitability for FRET analysis as well as functionality of fusions with SNAP- and HaloTag could be demonstrated by studying the transient interaction between chemotaxis response regulator CheY and its phosphatase CheZ.</p></div

Gateway-based B2H assay for detection of chaperone-effector interaction.

<p>Expression clones encoding reporter gene fusions GCN4-Zip N-terminal (T25-GCN4-Zip/T18-GCN4-Zip), GCN4-Zip C-terminal (GCN4-Zip-T25/GCN4-Zip-T18), unfused T18 and T25 fragments (without), SipA-InvB (InvB-T25/SipA-T18) and SipA_48–685-InvB (InvB-T25/SipA_48–685-T18) were co-expressed in the <i>cyaA</i>-deficient <i>E</i>. <i>coli</i> B2H reporter strain BTH101. Blue colonies indicate protein-protein interaction through T18/T25 CyaA fragment complementation.</p

Subcellular localization of chemotaxis proteins CheY and CheZ.

<p>Two representative <i>S</i>. Typhimurium cells co-expressing fusions of CheY and CheZ to Halo- and SNAP-Tag from Expression clone pWRG602 are shown. Fusion proteins were labeled with Atto655-coupled HaloTag ligand and the SNAP-tag ligand TMR-Star. Subcellular localization of protein clusters was visualized using super-resolution microscopy (dSTORM). Colocalization of the labeled fusion proteins was calculated in separate images using the Matlab-based software Slimfast with a distance threshold of 100 nm. The insets show autofluorescence of the bacteria after excitation at 488 nm which was used to determine their shapes (dashed white lines). Scale bars = 1 μm.</p

Strains used in this study.

<p>Strains used in this study.</p

Gateway-based split Gluc protein complementation assay (PCA) for detection of chaperone-effector interaction.

<p>Expression clones encoding InvB fused to Gluc^M43L₁₀₅ and SipA or a SipA variant lacking its CDB (SipA_48–685) fused to Gluc^M110L₁₀₆ or Gluc^M43L₁₀₅ and Gluc^M110L₁₀₆ alone (split Gluc) were transferred into <i>S</i>. Typhimurium and expression was induced with 50 ng*ml^-1 AHT. Gluc activity was quantified from intact cells (A) and bacterial lysates (B). The dotted line in (A) represents the background luminescence determined with an empty vector control (pWSK29). Mean and standard deviation out of three independent experiments done in triplicate is shown. Statistical analysis by Student’s <i>t</i>-test was done by comparing individual strains as depicted: ***, <i>P</i> < 0.001.</p

Overview of the system and plasmid combinations.

<p>Two Entry vectors suitable for C-terminal (left) or N-terminal (right) tagging are modified with genes of interest (GOI1 = red, GOI2 = green). The two resulting Entry clones are combined in a MultiSite Gateway LR reaction with an application-specific 3^rd Entry clone and Destination vector (below) resulting in the desired Expression clone (bottom). Plasmids pWRG-B2H-ENTR-N and pWRG-B2H-DEST-N allow for N-terminal tagging in bacterial two hybrid (B2H) applications if used together with compatible pWRG-ENTR-N1/2-derived Entry clones (right). Boxes represent the genes or gene fragments as indicated (<i>gluc</i>₁₀₅ = Gluc_M18–105, <i>gluc</i>₁₀₆ = Gluc_106–185, <i>scfp3a</i> = Super CFP 3a, <i>syfp2</i> = Super YFP 2, <i>snap</i> = SNAP-tag, <i>halo</i> = HaloTag 7, <i>t18</i> = T18 fragment of CyaA from <i>B</i>. <i>pertussis</i>, <i>t25</i> = T25 fragment of CyaA from <i>B</i>. <i>pertussis</i>), black rectangles symbolize stop-codons, tetracycline-inducible promoters are depicted as arrows and Ω stand for <i>rrnB</i>-derived terminators. Resistance cassettes, <i>att</i>R- and <i>att</i>L sites as well as by-products of the reactions are not shown.</p

Phosphomimetic Rac1 S71E induces filopodia formation.

<p>A) Treatment with 100 ng/ml EGF for 2 h induces pronounced formation of filopodia. Cells were stained for nuclei (DAPI, blue), actin cytoskeleton (rhodamin-phalloidin, red), and VASP (Alexa-488, green). B) HEp2 cells transfected with HA-tagged Rac1, Rac1 S71E, Cdc42, and Cdc42 S71E. Expression of GTPases was visualized by HA-staining, the actin cytoskeleton was stained with rhodamin-phalloidin. Only Rac1 S71E induced morphotype that is comparable with EGF-induced alterations. C) Phenotypes of HEp2 cells transfected with HA-tagged constitutive active mutants of Rac1 and Cdc42 as well as their phosphomimetic mutants S71E. Constitutively active (Q61L) Rac1 induced membrane ruffling whereas Rac1 S71E induced formation of filopodia. Filopodia formation is less pronounced in Cdc42 Q61L and Cdc42 Q61L/S71E transfected cells. Stained are nuclei (blue) and HA-tag (green); bar represents 10 µm. D) Active, GTP-bound form of Cdc42 was determined by G-LISA 24 h post transfection with constructs as indicated. Cdc42 Q61L was used for transfection experiments as positive control for experimental setup. Additionally, <i>C. difficile</i> toxin A (TcdA) was used as negative control for inactivation of Cdc42. The bar chart shows mean values ± SD of three (for TcdA) or four separate experiments.</p