Table 1.
Strains used in this study.
Fig 1.
Adaptation of the MultiSite Gateway Pro 3-fragment recombination for use in bacteria.
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 (PtetA). Transcription of fusion genes is stopped by a termination sequence derived from that of the rrnB operon (Ω).
Fig 2.
(A) Generation of Entry clones via BP reaction. The genes of interest (GOI, red) were amplified with flanking attB-sites and four terminal G’s (not shown). Depending on whether the GOIs should be fused N- or C-terminally to a reporter gene, the genes were amplified together with a ribosome binding site (RBS, turquoise) and no stop-codon (left) or without RBS and with stop-codon (right, black rectangle). Afterwards, the PCR products were recombined in a BP reaction with an appropriate pDONR vector yielding the desired Entry clones. (B) Generation of Entry clones via ‘classical’ cloning. The GOI is amplified with primers containing the flanking restriction enzyme recognition sites as indicated (bottom) and cloned in exchange with a ccdB-Cmr selection cassette into a custom-made Entry vector for C- (pWRG-ENTR-C1, left) or N-terminal (pWRG-ENTR-N1, right) reporter gene fusions, resulting in the desired Entry clone. Only the generation of attL1-attL4 Entry clones is shown. Construction of attL3-attL2 Entry clones follows the same principles, but alternative Entry- and Donor vectors and suitable primers have to be used (not shown).
Fig 3.
Overview of the system and plasmid combinations.
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 3rd 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 (gluc105 = GlucM18–105, gluc106 = Gluc106–185, scfp3a = Super CFP 3a, syfp2 = Super YFP 2, snap = SNAP-tag, halo = HaloTag 7, t18 = T18 fragment of CyaA from B. pertussis, t25 = T25 fragment of CyaA from B. pertussis), black rectangles symbolize stop-codons, tetracycline-inducible promoters are depicted as arrows and Ω stand for rrnB-derived terminators. Resistance cassettes, attR- and attL sites as well as by-products of the reactions are not shown.
Fig 4.
Gateway-based B2H assay for detection of chaperone-effector interaction.
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 SipA48–685-InvB (InvB-T25/SipA48–685-T18) were co-expressed in the cyaA-deficient E. coli B2H reporter strain BTH101. Blue colonies indicate protein-protein interaction through T18/T25 CyaA fragment complementation.
Fig 5.
Gateway-based split Gluc protein complementation assay (PCA) for detection of chaperone-effector interaction.
Expression clones encoding InvB fused to GlucM43L105 and SipA or a SipA variant lacking its CDB (SipA48–685) fused to GlucM110L106 or GlucM43L105 and GlucM110L106 alone (split Gluc) were transferred into S. 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 t-test was done by comparing individual strains as depicted: ***, P < 0.001.
Fig 6.
Localization and interaction of chemotaxis proteins CheY and CheZ.
(A) Fluorescence microscopy of S. Typhimurium MvP103 (ΔsseC) cells co-expressing CheY-SYFP2 and CheZ-SCFP3a fusion proteins from Expression clone pWRG415. Signals from CFP- and YFP channels are shown separately as well as merged and pseudo-colored as indicated. Both fusion proteins formed intense clusters at the poles of the bacterial cells. The product of the difference from the mean (PDM) of both channels was calculated and plotted to show sites of complex formation (orange). The shapes of individual bacteria were illustrated by dashed white lines. Scale bar = 1 μm, Scale in PDM image = positive covariance (orange) and negative covariance (blue) (B) The same Expression clone as in (A) was used to detect the dynamics of the CheY-Z interaction by FRET in E. coli VS104 [Δ(cheY-cheZ)] (left) and S. Typhimurium MvP103 (right) cells. The fluorescence intensities of the CFP (green) and YFP (red) channels are depicted in the upper graphs. The YFP/CFP ratios calculated from the fluorescence intensities are shown below. Addition (black arrows) and removal (blue arrows) of the chemotactic attractant MeAsp resulted in a decrease (no interaction) or increase (interaction) of the YFP/CFP quotient, respectively.
Fig 7.
Subcellular localization of chemotaxis proteins CheY and CheZ.
Two representative S. 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.