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J Bacteriol, May 1998, p. 2434-2441, Vol. 180, No. 9
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The Prosequence of Pro-sigma K Promotes Membrane Association and Inhibits RNA Polymerase Core Binding

Bin Zhang,1 Antje Hofmeister,2 and Lee Kroos1,*

Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824,1 and Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 021382

Received 10 November 1997/Accepted 22 January 1998

Pro-sigma K is the inactive precursor of sigma K, a mother cell-specific sigma factor responsible for the transcription of late sporulation genes of Bacillus subtilis. Upon subcellular fractionation, the majority of the pro-sigma K was present in the membrane fraction. The rest of the pro-sigma K was in a large complex that did not contain RNA polymerase core subunits. In contrast, the majority of the sigma K was associated with core RNA polymerase. Virtually identical fractionation properties were observed when pro-sigma E was analyzed. Pro-sigma K was completely solubilized from the membrane fraction and the large complex by Triton X-100 and was partially solubilized from the membrane fraction by NaCl and KSCN. The membrane association of pro-sigma K did not require spoIVF gene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-sigma K processing in the mother cell. Furthermore, pro-sigma K associated with the membrane when overproduced in vegetative cells. Overproduction of pro-sigma K in sporulating cells resulted in more pro-sigma K in the membrane fraction. In agreement with the results of cell fractionation experiments, immunofluorescence microscopy showed that pro-sigma K was localized to the mother cell membranes that surround the mother cell and the forespore in sporulating wild-type cells and mutant cells that do not process pro-sigma K. Treatment of extracts with 0.6 M KCl appeared to free most of the pro-sigma K and sigma K from other cell constituents. After salt removal, sigma K, but not pro-sigma K, reassociated with exogenous core RNA polymerase to form holoenzyme. These results suggest that the prosequence inhibits RNA polymerase core binding and targets pro-sigma K to the membrane, where it may interact with the processing machinery.

* Corresponding author. Mailing address: Department of Biochemistry, Michigan State University, East Lansing, MI 48824. Phone: (517) 355-9726. Fax: (517) 353-9334. E-mail: kroos{at}pilot.msu.edu.

J Bacteriol, May 1998, p. 2434-2441, Vol. 180, No. 9
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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