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Applied and Environmental Microbiology, May 2002, p. 2624-2628, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2624-2628.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Marcus Kaltwasser, Thomas Wiegert, and Wolfgang Schumann*

Institute of Genetics, University of Bayreuth, D-95445 Bayreuth, Germany

Received 3 December 2001/ Accepted 19 February 2002

Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3' terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein+, yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.

* 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.

Applied and Environmental Microbiology, May 2002, p. 2624-2628, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2624-2628.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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