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Journal of Bacteriology, April 1999, p. 2346-2350, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Replacement of Vegetative sigma A by Sporulation-Specific sigma F as a Component of the RNA Polymerase Holoenzyme in Sporulating Bacillus subtilis

Matthew Lord,dagger Daniela Barillà,Dagger and Michael D. Yudkin*

Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

Received 3 December 1998/Accepted 8 February 1999

Soon after asymmetric septation in sporulating Bacillus subtilis cells, sigma F is liberated in the prespore from inhibition by SpoIIAB. To initiate transcription from its cognate promoters, sigma F must compete with sigma A, the housekeeping sigma factor in the predivisional cell, for binding to core RNA polymerase (E). To estimate the relative affinity of E for sigma A and sigma F, we made separate mixtures of E with each of the two sigma factors, allowed reconstitution of the holoenzyme, and measured the concentration of free E remaining in each mixture. The affinity of E for sigma F was found to be about 25-fold lower than that for sigma A. We used quantitative Western blotting to estimate the concentrations of E, sigma A, and sigma F in sporulating cells. The cellular concentrations of E and sigma A were both about 7.5 µM, and neither changed significantly during the first 3 h of sporulation. The concentration of sigma F was extremely low at the beginning of sporulation, but it rose rapidly to a peak after about 2 h. At its peak, the concentration of sigma F was some twofold higher than that of sigma A. This difference in concentration cannot adequately account for the replacement of sigma A holoenzyme by sigma F holoenzyme in the prespore, and it seems that some further mechanism---perhaps the synthesis or activation of an anti-sigma A factor---must be responsible for this replacement.

* Corresponding author. Mailing address: Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom. Phone: 44 1865 275302. Fax: 44 1865 275297. E-mail: mdy{at}bioch.ox.ac.uk.

dagger Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.

Dagger Present address: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

Journal of Bacteriology, April 1999, p. 2346-2350, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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