InsTipα insertion sites and expression of the resulting InsTip1 fusion proteins. (A to E) Results for the five strains with InsTipα insertions in (A) atpD, (B) ppiD, (C) yraI, (D) yicO, and (E) yfdX. Top panels, schematic depiction of the insertion site and orientation of InsTipα into each target gene. The respective gene is represented by a black arrow and its designation, and its total length is indicated in bp. InsTipα is drawn schematically above the open reading frame, and its insertion site is indicated by a dotted line with the exact position of the nucleotide preceding the inserted sequence. Bottom panels, growth curves (filled squares) and β-Gal activities (filled bars) of WH207(λtet50)/pWH1911 and the respective candidate strains (open squares and open bars) grown at 37°C in MacConkey medium without indicator. Growth is expressed by the optical density at 600 nm (OD₆₀₀) on the left y axis. The right y axis displays the β-Gal activities in Miller units (MU) (23). Each value was determined in triplicate, and the standard deviations are shown (error bars).

InsTipα architecture, in vivo screen, and in vivo activity. (A) Architecture of the insertion element InsTipα. Triangles represent the ME for transposase binding, the white box indicates the linker element, and the gray box encodes Tip. The ochre stop codon is symbolized by “S.” The white and black arrows represent the kanamycin resistance gene (aphAIII) and its promoter, and a lollipop represents the bidirectional transcriptional terminator from Tn10 (30). The resistance cassette is flanked by two Flp recombinase target sites (FRT) drawn as black arrows. The amino acid sequence encoded by the mosaic element at the leftward 5′ end, the linker, and tip is shown in single-letter abbreviations above the nucleotide sequence that is displayed in white type on a black background. The stop codon is marked by an asterisk. (B and C) In vivo screening system for TetR-inducing InsTip1 fusions. (B) Screening was carried out with E. coli WH207(λtet50) containing a chromosomally encoded tetA-lacZ transcriptional fusion. TetR (gray ovals) binds tetO and represses lacZ. (C) Integration of the insertion element InsTipα into an expressed open reading frame (geneX) results in a GeneX-InsTip1 fusion protein (light gray oval with X). Induction of TetR by this fusion protein is indicated by β-Gal activity. (D) In vivo activity of the TrxA-InsTip1 fusion. The leftmost pair of bars shows the repressed (white) and tetracycline-induced (gray) β-Gal activities. The next seven bars show the activities of the TrxA-Tip fusion control without (white) and with IPTG at the indicated concentrations (black; 15, 30, 60, 125, 250, and 500 μM). The right group of bars shows the β-Gal activities obtained with the TrxA-InsTip1 fusion under the same conditions. The y axis displays the β-Gal activities in Miller units (MU) (23). (E) Western blot of 10 μg of crude cell lysates separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed with an anti-thioredoxin A monoclonal antibody (Invitrogen).

Carbon source-dependent differential expression of PpiD-InsTip1. β-Gal activities of WH207(λtet50)/pWH1911 (closed bars) and WH852 (open bars) grown at 37°C to an optical density at 600 nm of 0.2 in M9 minimal medium supplemented with 50 mM glycerol or pyruvate. The y axis displays the β-Gal activities in Miller units (MU) (23). Each value was determined in triplicate and is shown with standard deviations (error bars). The dotted lines indicate the background β-Gal activities determined for the untagged parent strain, which were subtracted from the β-Gal activities of the tagged strain to determine the induction factor.