Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for Bacillus subtilis

Here we describe the construction and application of six newtagging vectors allowing the fusion of two different types oftagging sequences, epitope and localization tags, to any Bacillussubtilis protein. These vectors are based on the backbone ofpMUTIN2 and replace the lacZ gene with tagging sequences. Fusionof the tagging sequences occurs by PCR amplification of the3' terminal part of the gene of interest (about 300 bp), insertioninto the tagging vector in such a way that a fusion proteinwill be synthesized upon integration of the whole vector viahomologous recombination with the chromosomal gene. Three ofthese tagging sequences (FLAG, hemagglutinin, and c-Myc) allowthe covalent addition of a short epitope tag and thereby detectionof the fusion proteins in immunoblots, while three other tags(green fluorescent protein⁺, yellow fluorescent protein, andcyan fluorescent protein) are helpful in assigning proteinswithin one of the compartments of the cell. The versatilityof these vectors was demonstrated by fusing these tags to thecytoplasmically located HtpG and the inner membrane proteinFtsH.

INTRODUCTION

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Introduction

Sequencing the Bacillus subtilis genome has revealed about 4,100genes, the function of which approximately 60% has been identifiedeither experimentally or by computer-based analysis (9). Asa first step to elucidate the function of the remaining $~$ 1,600genes, a network of 18 European and 12 Japanese laboratorieshas systematically inactivated most of these genes of unknownfunction (17). To obtain this goal, the integration vector pMUTINwas constructed (18). This vector is unable to replicate inB. subtilis, but upon insertion of about 300 bp derived fromthe coding region of the gene to be inactivated by PCR, it canintegrate into that gene by homologous recombination. Integrationof the recombinant pMUTIN vector into the target gene transcriptionallyfuses lacZ to that promoter of the gene, and downstream genescan be controlled by the isopropyl-ß-D-thiogalactopyranoside(IPTG)-dependent Pspac promoter (18).

A further characterization of unknown gene products at the proteinlevel requires their detection by antibodies. Production ofantibodies requires purification of the protein followed byimmunization of an animal, normally a rabbit. This procedureis time consuming and expensive, and the antibodies obtainedoften vary in their quality. To circumvent these problems, themethod of choice is the use of epitope-tagging vectors and/orgreen fluorescent protein (GFP) fusions, both of which are importanttools in eukaryotic systems (1, 12). While epitope-tagging vectorshave never been described for B. subtilis, GFP fusion vectorsare available, but they do not allow fusion of the localizationtag to chromosomally located genes (4, 10, 11). We made useof the pMUTIN vector and modified it in such a way to allowa translational fusion of the three epitope tags FLAG, hemagglutinin(HA), and c-Myc and the localization tags GFP, cyan fluorescentprotein (CFP), and yellow fluorescent protein (YFP) to the 3'end of any gene within the B. subtilis chromosome and of anyother bacterial species not allowing replication of pMUTIN.While the FLAG tag is an artificial 8-amino-acid residue-longpeptide (7), c-Myc (10 amino acid residues) and HA (9 aminoacid residues) were derived from the human c-myc proto-oncogeneand the HA of the influenza virus, respectively (3, 20). Antibodiesspecifically recognizing these tags are commercially available.GFP and its two variants are highly useful fluorescent tagsfor studying the localization and dynamics of proteins in livingcells.

Construction of six tagging integration vectors

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Construction of six tagging...

We started from the integration vector pDE01, a precursor ofthe pMUTIN2 derivative that carries bgaB instead of the lacZreporter gene (E. Deuerling, unpublished work); this gene codesfor a heat-stable ß-galactosidase (5). First, theSalI-SphI fragment carrying the bgaB gene was replaced witha polylinker with several unique restriction enzyme sites (Table1), resulting in the plasmid pMUTIN-Poly and thereby destroyingthe SalI site. To ensure efficient termination of transcriptionimmediately downstream of the hybrid gene, the trpA terminatorof the Escherichia coli tryptophan operon (8), assembled fromtwo complementary oligonucleotides (Table 1), was inserted intothe SpeI-SphI sites of pMUTIN-Poly in such a way that the SphIsite was destroyed, resulting in pMUTIN-Ter. This vector servedas a backbone for the insertion of the six tagging sequences.

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TABLE 1. Complementary oligonucleotides used to create short insertions into the integration vector

The DNA sequences coding for the three different epitopes (Table1) were ligated as complementary oligonucleotides into the XmaIIIand SpeI sites, resulting in pMUTIN-FLAG, -cMyc, and -HA (Fig.1A); by this manipulation the SpeI site was destroyed in allthree vectors. The codons for the different amino acids weredesigned on the basis of the codon usage table published forB. subtilis (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?=Bacillus+subtilis+[gbbct]),and the open reading frames were terminated with two consecutivestop codons to ensure efficient termination of translation (Table1). The correct DNA sequences of all three epitope tags wereconfirmed by DNA sequencing. The coding regions for the GFPand its two variants were generated by PCR and flanked withXmaIII and SpeI sites (Table 2). As templates, we used the plasmidspMN402 (coding for GFP⁺, a variant of the wild-type gfp exhibitingincreased fluorescence [14] [using oligonucleotides ON1 andON2] [Table 2]) and pSG1186 and pSG1187 (ON3 and ON4, codingfor cfp and yfp, respectively) (4), resulting in the three plasmidspMUTIN-GFP⁺, -CFP, and -YFP (Fig. 1B). All six tagging vectorsretain the unique restriction sites KpnI, Eco47-3, ClaI, andXmaIII for insertion of PCR fragments.

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FIG. 1. Tagging integration vectors. (A) Epitope-tagging vectors carrying the coding sequences for the three epitopes FLAG, c-Myc, and HA. (B) Localization-tagging vectors containing the coding sequences for gfp⁺, yfp, and cfp. All vectors are derivatives of pBR322 and do not replicate in B. subtilis.

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TABLE 2. Oligonucleotides used in this study

Construction of recombinant tagging vectors

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Construction of recombinant...

To prove that these vectors function appropriately, we decidedto fuse the tagging sequences to two different genes, one codingfor the cytoplasmic heat shock protein HtpG and the other forthe ATP- and Zn²⁺-dependent protease FtsH, which is anchoredto the cytoplasmic membrane (2, 16). To accomplish this goal,about 300 bp from the 3' end of both genes excluding their stopcodon was amplified by PCR using the oligonucleotides ON5 andON6 in the case of htpG and ON7 and ON8 for ftsH (Table 2).Both PCR products were cleaved with KpnI and XmaIII and ligatedinto the six tagging vectors treated with the same enzymes.In the last step, the 12 recombinant plasmids were used to transformcompetent B. subtilis strain 1012 cells (13). Transformantswere selected on Luria-Bertani plates containing erythromycinand were further analyzed by PCR for integration of one copyeach of the plasmid at the correct locus (data not shown); onestrain each was used in the following experiments.

Fusion proteins carrying the three epitope tags can be detected using specific antibodies

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Fusion proteins carrying the...

In the next step, we tested for the production of fusion proteinscarrying the different epitope tags. Cells carrying htpG orftsH fused to either the FLAG, c-Myc, or HA epitope were analyzedfor the presence of the appropriate fusions. As can be seenin Fig. 2A, ${alpha}$ HtpG cross-reacted with a protein of about 72 kDapresent in all strains tested with the exception of the htpGknockout. The same result was obtained for FtsH, which couldbe detected in all strains but the ftsH knockout (Fig. 2B).When antibodies raised against the three epitopes were usedto probe the extracts, they specifically detected the two fusionproteins containing FLAG (Fig. 2C), c-Myc (Fig. 2D), and HA(Fig. 2E). We conclude from these results that all three integrationvectors function as expected and can be used to add one of thethree epitopes to either a cytoplasmic or an integral membraneprotein.

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FIG. 2. Detection of the two proteins HtpG and FtsH by immunoblot analysis. B. subtilis strain 1012 and its isogenic derivatives were grown at 37°C to mid-exponential growth phase, and sample preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and for immunoblot analysis was performed as described previously (6). An equal amount of protein was applied per lane. The blots were probed with ${alpha}$ HtpG (A), ${alpha}$ FtsH (B), ${alpha}$ FLAG (C), ${alpha}$ c-Myc (D), and ${alpha}$ HA (E). The bands representing the tagged proteins are marked by an arrow. Antibodies against HtpG and FtsH have been raised against purified His-tagged proteins (2, 16). Antibodies raised against the epitope tags were purchased from Sigma ( ${alpha}$ FLAG and ${alpha}$ c-Myc, clone 9E10) and Roche Diagnostics ( ${alpha}$ HA, clone 12CA5).

Fusion proteins carrying the three GFP variants can be demonstrated using anti-GFP antibodies

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Next, we wanted to detect the six fusion proteins carrying thelocalization tags at their C termini by immunoblotting. Again,the proteins of the different cell extracts were separated byelectrophoresis, blotted, and probed with three different antibodies.When the blot was treated with anti-HtpG ( ${alpha}$ HtpG), cross-reactingmaterial was detected in all cell lysates, with the exceptionof the ${Delta}$ htpG strain (Fig. 3A). Cells from strains expressinga tagged HtpG protein exhibited a band which by its molecularmass represented the fusion protein. Two additional bands canbe visualized, one with the molecular mass of HtpG and the secondmigrating between the two others (Fig. 3A), both most probablyrepresenting degradation products. A similar observation wasmade when the blot was probed with ${alpha}$ FtsH. Cross-reactive materialwas present in all extracts but those from the ${Delta}$ ftsH strain (Fig.3B). Again, strains producing the tagged FtsH exhibited twoadditional bands, one major with the molecular mass of FtsHand one minor migrating between the two others (Fig. 3B). Thesedata suggest that the fusion proteins are a target for one ormore proteases within the B. subtilis cells. Furthermore, theantibodies reacted with a protein of about 50 kDa, not presentin the ftsH knockout and therefore representing a degradationproduct of FtsH. When ${alpha}$ GFP antibodies were used, they detectedthe fusion protein only and another protein present in all extracts(Fig. 3C).

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FIG. 3. Detection of HtpG and FtsH fusion proteins by immunoblot analysis. Preparation of the immunoblots followed the procedure described in the Fig. 2 legend. The blots were probed with ${alpha}$ HtpG, ${alpha}$ FtsH, and ${alpha}$ GFP. The bands representing the tagged proteins are marked by an arrow. ${alpha}$ GFP was obtained from Clontech.

Since the tagged proteins could be directly visualized usingfluorescence microscopy only in the case of the GFP⁺ versions(only filter sets for GFP were available; data not shown) andsince descriptions of cells expressing FtsH-GFP have alreadybeen published (19), we decided to measure the fluorescenceof all three versions using a fluorometer. We chose the threestrains producing HtpG fusion proteins, allowing the inductionof its gene by heat shock (15). It turned out that only HtpG-GFP⁺could be detected in the absence of heat stress (Table 3), whileall three fusion proteins were clearly present after a heatshock. This experiment independently confirms that all threeproteins are indeed produced.

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TABLE 3. Fluorescence measurements in cells synthesizing HtpG labeled with three different GFP tags

Conclusions

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Conclusions

(i) Six tagging integration vectors were constructed allowingtranslational fusions of two types of tagging sequences, epitope,and localization, to the 3' end of any chromosomal gene of interest.If this gene is part of a polycistronic operon and if one ofthe downstream genes is essential, its transcription can beensured by an IPTG-regulable promoter. These vector plasmidswere primarily constructed for use in B. subtilis but can beapplied to any bacterial species not allowing replication ofthe pBR322-based plasmids. But expression of downstream genesdepends on synthesis of the LacI repressor protein and properfunctioning of the Pspac promoter. (ii) The epitope sequences(FLAG, c-Myc, and HA) allow detection of tagged proteins withinthe cell using commercially available antibodies. Furthermore,the FLAG and HA tags can be used to purify the fusion proteinsby affinity chromatography. (iii) The localization tags GFP⁺,which produces enhanced fluorescence; YFP; and CFP can be usedto localize a protein to a specific compartment within the cell.(iv) To prove that all six tagging vectors are functional, theywere fused to two different genes, one coding for the cytoplasmicprotein HtpG and the other for the integral membrane proteinFtsH. All 12 fusion proteins could be detected using specificantibodies. (v) The six plasmids can be ordered from the BacillusGenetic Stock Center (http://bacillus.biosci.ohio-state.edu).The DNA sequences of the plasmids and their plasmid maps canbe found at http://btbgn1.bio.uni-bayreuth.de/lsgenetik1/frames.htm.

ACKNOWLEDGMENTS

We thank Peter J. Lewis for providing plasmids pSG1186 and pSG1187.

Financial support was provided by EU project QLRT-1999-00413and by the Fonds der Chemischen Industrie.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Genetics, University of Bayreuth, D-95445 Bayreuth, Germany. Phone: 49 (921) 552708. Fax: 49 (921) 552710. E-mail: wolfgang.schumann{at}uni-bayreuth.de.

REFERENCES

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