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Journal of Bacteriology, July 2001, p. 3885-3889, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.3885-3889.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

SsrA-Mediated Tagging in Bacillus subtilis

Thomas Wiegert and Wolfgang Schumann*

Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany

Received 11 January 2001/Accepted 4 April 2001

A general mechanism in bacteria to rescue stalled ribosomes involves a stable RNA encoded by the ssrA gene. This RNA, termed tmRNA, encodes a proteolytic peptide tag which is cotranslationally added to truncated polypeptides, thereby targeting them for rapid proteolysis. To study this ssrA-mediated mechanism in Bacillus subtilis, a bipartite detection system was constructed that was composed of the HrcA transcriptional repressor and the bgaB reporter gene coding for a heat-stable beta -galactosidase fused to an HrcA-controlled promoter. After the predicted proteolysis tag was fused to HrcA, the reporter beta -galactosidase was expressed constitutively at a high level due to the instability of the tagged HrcA. Replacement of the two C-terminal alanine residues of the tag by aspartate rendered the repressor stable. Replacement of the hrcA stop codon by a transcriptional terminator sequence rendered the protein unstable; this was caused by trans translational addition of the proteolytic tag. Inactivating the B. subtilis ssrA or smpB (yvaI) gene prevented the trans translational tagging reaction. Various protease-deficient strains of B. subtilis were tested for proteolysis of tagged HrcA. HrcA remained stable only in clpX or clpP knockouts, which suggests that this ATP-dependent protease is primarily responsible for the degradation of SsrA-tagged proteins in B. subtilis.

* Corresponding author. Mailing address: Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany. Phone: (49)921 552708. Fax: (49)921 552710. E-mail: wolfgang.schumann{at}unibayreuth.de.

Journal of Bacteriology, July 2001, p. 3885-3889, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.3885-3889.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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