Random Insertion of a TetR-Inducing Peptide Tag into Escherichia coli Proteins Allows Analysis of Protein Levels by Induction of Reporter Gene Expression

The insertion element InsTip ${alpha}$ was constructed to generate proteinexpression data. It randomly fuses the TetR-inducing peptideTip to the affected reading frame. Fusion protein expressionis quantified by Tet-regulated reporter gene expression. Theexpression patterns of tagged Escherichia coli genes fully agreewith published data from transcriptional fusions or microarrays,validating the Tip tag approach.

INTRODUCTION

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Introduction

Proteome expression profiles provide important information butare cumbersome to obtain as the data are usually collected bytwo-dimensional electrophoresis (9, 25). When genome sequencesare available, proteome-wide sequence-directed tagging approachesare an alternative (7, 13, 33). If directed tagging is not possible,random transposon-mediated tagging approaches are employed tofuse large reporter genes like lacZ to reading frames. The expressionof these large reporter fusions is indicative of the amountof originally encoded protein expressed under the particularexperimental conditions (21, 28). However, large fusion proteinsmay exhibit different properties than the untagged proteins,e.g., by affecting their solubility (19) or localization throughsteric hindrance (13). We describe here an approach to obtaina genetic readout of proteomic information by creating fusionswith a short hexadecapeptide, called Tip (transcription-inducingpeptide), that induces Tet repressor (16). For that purposewe constructed an insertion element, called InsTip ${alpha}$ , to generatetransposome-mediated random (12) in-frame translational fusionswith Tip in Escherichia coli. We demonstrate here that thiselement can be used to determine the expression characteristicsof proteins in five such fusion mutants.

Construction and function of InsTip ${alpha}$ in E. coli.

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Construction and Function of...

We assembled the 1,816-bp-long insertion element InsTip ${alpha}$ (Fig.1A) from previously published vectors and oligonucleotides,amplified it by PCR, and cloned it into the PvuII-digested vectorpWH1866 (17) (see Table 1 for a list of all bacterial strains,plasmids, and phages) to obtain pWH1912. (Construction detailsand sequences of all plasmids generated in this study are availableupon request.) InsTip ${alpha}$ is flanked by 19-bp-long mosaic elements(ME) (26), the binding sites of the hyperactive transposaseTn5 (8). The leftward end of InsTip ${alpha}$ (Fig. 1A) contains a sequencedesigned to fuse a tag, termed InsTip1, to any protein encodedat the insertion site. An open reading frame spans the ME encoding7 amino acids, followed by a sequence encoding a 7-amino-acidlinker and the 16-residue Tip element terminated by an ochrestop codon. The insertion element also contains aphAIII, whichconfers kanamycin resistance in gram-negative and -positivebacteria (10), flanked by two Flp recombinase target sites toallow excision of the antibiotic resistance gene (4), therebyminimizing genomic interference.

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FIG. 1. InsTip ${alpha}$ architecture, in vivo screen, and in vivo activity. (A) Architecture of the insertion element InsTip ${alpha}$ . 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( ${lambda}$ tet50) containing a chromosomally encoded tetA-lacZ transcriptional fusion. TetR (gray ovals) binds tetO and represses lacZ. (C) Integration of the insertion element InsTip ${alpha}$ 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 ß-Gal activity. (D) In vivo activity of the TrxA-InsTip1 fusion. The leftmost pair of bars shows the repressed (white) and tetracycline-induced (gray) ß-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 ß-Gal activities obtained with the TrxA-InsTip1 fusion under the same conditions. The y axis displays the ß-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).

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TABLE 1. Bacterial strains, plasmids, and phage used in this study

To determine if InsTip ${alpha}$ insertion into an open reading framecan generate a protein fusion that induces TetR, InsTip ${alpha}$ wasfused to TrxA of E. coli, which had previously been used toexpress and characterize Tip (16). The constructed plasmid,pWH1909, expressing TrxA-InsTip1 under P_tac control, and theplasmid pWH1911, expressing TetR constitutively at a low level,were cotransformed into E. coli WH207( ${lambda}$ tet50) (35). The additionof IPTG (isopropyl-ß-D-thiogalactopyranoside) to theresulting strain leads to expression of the InsTip1 fusion protein,and subsequent induction of TetR should relieve repression oflacZ (Fig. 1C). We titrated the strain with IPTG concentrationsof 15 to 500 µM to express increasing amounts of TrxA-InsTip1(Fig. 1D). Plasmid pWH2101 expressing TrxA-Tip was cotransformedinstead of pWH1909 to provide a positive control for induction(16). A plateau of ß-galactosidase (ß-Gal)activity was obtained for concentrations above 60 µM ofIPTG with both constructs (Fig. 1D). Western blot analyses carriedout simultaneously demonstrated nearly identical expressionlevels of the fusion proteins TrxA-Tip and TrxA-InsTip1 (Fig.1E). Thus, neither the length nor the primary structure of thepeptide segment encoded by the ME and linker affects the TetR-inducingproperties of Tip.

Purified hyperactive transposase and PvuII-restricted, gel-purifiedInsTip ${alpha}$ DNA were incubated to form transposomes (2) and electroporated(8) into E. coli WH207( ${lambda}$ tet50)/pWH1911, resulting in 6,400 kanamycin-resistantcandidates on MacConkey agar plates. After 36 h of incubationat 37°C, 5 colonies displayed a yellow color indicatinginduction of TetR. Chromosomal DNA from the five strains wasprepared and sequenced to determine their InsTip ${alpha}$ integrationsites (2). The diverse sequences obtained at the InsTip ${alpha}$ integrationloci agree in indicating its random insertion (12). Table 2lists the strains obtained and the genes with their respectiveInsTip ${alpha}$ insertion sites (see also Fig. 2), together with informationabout the function and subcellular localization of the affectedproteins.

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TABLE 2. Annotation of InsTip ${alpha}$ insertion strains

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FIG. 2. InsTip ${alpha}$ insertion sites and expression of the resulting InsTip1 fusion proteins. (A to E) Results for the five strains with InsTip ${alpha}$ insertions in (A) atpD, (B) ppiD, (C) yraI, (D) yicO, and (E) yfdX. Top panels, schematic depiction of the insertion site and orientation of InsTip ${alpha}$ into each target gene. The respective gene is represented by a black arrow and its designation, and its total length is indicated in bp. InsTip ${alpha}$ 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 ß-Gal activities (filled bars) of WH207( ${lambda}$ tet50)/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 ß-Gal activities in Miller units (MU) (23). Each value was determined in triplicate, and the standard deviations are shown (error bars).

Determining fusion protein expression by InsTip1-induced ß-Gal expression.

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Determining fusion protein...

We first examined the growth curves of the five candidates andtheir parent strain WH207( ${lambda}$ tet50)/pWH1911 at 37°C in MacConkeymedium lacking the indicator bromocresol purple (Fig. 2A to E).The growth rates of all strains were identical (Fig. 2B to E),except that for WH851, which was clearly growth defective (Fig.2A). We assumed that the InsTip ${alpha}$ insertion in atpD inactivatesATP synthase (29). We therefore analyzed the growth of WH851and the atpD⁺ parent strain on minimal medium with a nonfermentablecarbon source. In contrast to the atpD⁺ strain, WH851 displayedno growth on agar plates containing minimal medium with succinateas the sole carbon source (data not shown). Since growth onthis medium depends on a functional oxidative phosphorylationsystem, this phenotype is consistent with a nonfunctional ATPsynthase (14, 18). This assumption was further supported bythe growth phenotype in liquid minimal medium supplemented withlimiting amounts of glucose as the sole carbon source (18, 34);in this medium, WH851 reached a maximal optical density of 0.3,in contrast to an optical density of 1.0 for the atpD⁺ referencestrain (data not shown), again indicating the absence of oxidativephosphorylation.

At regular intervals during the growth periods, cells were collectedfrom all candidate strains and the parent strain to determinethe ß-Gal activities during exponential and stationarygrowth (Fig. 2A to E). The parent strain showed no change inß-Gal activity throughout the entire experiment, althoughthe ß-Gal activity could be induced 40-fold by theaddition of Tc (Fig. 1D) (35). Only background levels of ß-Galactivity were detected for the candidate strains during exponentialgrowth (Fig. 2A to E). However, for PpiD-InsTip1, YraI-InsTip1,YicO-InsTip1, and YfdX-InsTip1, a rapid increase in ß-Galactivity was observed at the onset of the stationary growthphase, reaching a plateau by the 20-h time point of the experiment(Fig. 2B to E). With a value of approximately 1,100 units (23),WH852 has demonstrated the highest level of ß-Galactivity. In contrast, we detected an increase in ß-Galactivity within the stationary growth phase for WH851, but itwas delayed and not as pronounced as the increases for the otherstrains. In addition, it did not reach a plateau during the40-h time frame of the experiment (Fig. 2A).

We calculated an approximately sixfold increase in ß-Galactivity of WH853 with the YraI-InsTip1 fusion for the transitionfrom mid-exponential growth (4 h) to full stationary phase (20to 40 h) (Fig. 2C). This observation is in excellent agreementwith published data from transcriptome profiling in which a4.1-fold increase in the mRNA level was observed due to thegrowth phase shift (11).

In WH854, we established expression of the YicO-InsTip1 fusionin stationary phase. yicO promoter activity was previously reportedto be induced in the early stationary phase by overexpressionof the two-component regulatory system BaeSR using a transcriptionalreporter gene fusion (1). In contrast, a whole-genome microarrayanalysis of BaeSR-dependent gene expression from mid-exponential-phasecultures showed no increase of yicO expression (24). Our resultsfor YicO-InsTip1 expression are in full agreement with the resultsof both of these earlier reports and explain their apparentlydiverse results.

In WH855, InsTip ${alpha}$ had integrated into the yfdX gene. Since yfdXis an essential gene (6), both the insertion and the identicalgrowth curves of WH855 and the yfdX⁺ reference strain at 37°Cindicate that residues in a C-terminal position from Gly158are not needed for growth under these conditions (Fig. 2E).A transcriptome comparison of exponentially grown cells withstationary-phase cells of E. coli showed that yfdX is inducedsignificantly in the stationary phase (see supplemental datafrom reference 31 as cited in reference 22). Our data from theYfdX-InsTip1 fusion corroborate this result (Fig. 2E).

Besides being able to detect the expression of InsTip1 fusionproteins on MacConkey and minimal media, we can also use InsTip1to determine quantitative differences in protein expression.A threefold-higher expression level of a ppiD-lacZ transcriptionalfusion has been reported for exponentially growing E. coli inminimal medium with pyruvate as the sole carbon source thanwith glycerol as the sole carbon source (5). Therefore, we analyzedß-Gal activity of WH852 cells under these conditionsand found a twofold-higher ß-Gal activity for growthwith pyruvate than with glycerol within the exponential growthphase. Thus, the result obtained with this translational fusionis in excellent agreement with the data from a transcriptionalfusion (Fig. 3).

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FIG. 3. Carbon source-dependent differential expression of PpiD-InsTip1. ß-Gal activities of WH207( ${lambda}$ tet50)/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 ß-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 ß-Gal activities determined for the untagged parent strain, which were subtracted from the ß-Gal activities of the tagged strain to determine the induction factor.

Conclusions.

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Conclusions.

We have successfully used the peptide Tip (16) as a proteinexpression tag. We constructed an in-frame fusion which indicatesthe expression of the tagged target gene by readout of a separatedlacZ gene. In contrast to direct lacZ fusions, this introducesonly a 30-amino-acid-long extension after the insertion site,turning the protein fusion into an inducer for TetR. The inductionof a separated reporter gene should lead to amplification ofthe signal. We constructed an InsTip ${alpha}$ insertion element, formedthe complex with hyperactive tranposase (8), randomly mutated(12) the model organism E. coli, and obtained five candidatesout of 6,400 insertion mutants. The tagged proteins were detectedduring the stationary growth phase, extending and confirmingpublished expression data from transcriptional fusions or microarraysfor atpD, ppiD, yraI, yicO, and yfdX, thereby validating theTip tag approach. We could also measure expression of ppiD duringthe exponential growth phase. Together with the ability to detectproteins of essential and nonessential genes, as well as proteinslocalized in the cytoplasm, the inner membrane, or the periplasmwith the tag, our results clearly demonstrate the applicabilityof InsTip ${alpha}$ as a global tag. These results encourage us to furtherimprove the insertion efficiency and to expand the detectionof active InsTip1 fusion proteins by avoiding polar effectson downstream genes through inclusion of an outward promoterin the element (15). We also aim to improve detection of proteinexpression within the exponential phase via enhanced screening,for example, by reduction of the relatively high basal levelof ß-Gal activity under repressed conditions to enlargethe detection window (20) or by the use of selection systems.

ACKNOWLEDGMENTS

This work was supported by the Deutsche Forschungsgemeinschaftthrough SFB473 and GRK805 and by the Fonds der Chemischen Industrie.

We thank Marcus Klotzsche and Anke Friedlein for fruitful discussions.

FOOTNOTES

* Corresponding author. Mailing address: Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany. Phone: 49-9131-85-28081. Fax: 49-9131-85-28082. E-mail: whillen{at}biologie.uni-erlangen.de.

REFERENCES

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