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Journal of Bacteriology, June 2001, p. 3623-3630, Vol. 183, No. 12
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.12.3623-3630.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Analysis of Promoter Recognition In Vivo Directed by sigma F of Bacillus subtilis by Using Random-Sequence Oligonucleotides

Edward Amaya,dagger Anastasia Khvorova,Dagger and Patrick J. Piggot*

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Received 27 December 2000/Accepted 26 March 2001

Formation of spores from vegetative bacteria by Bacillus subtilis is a primitive system of cell differentiation. Critical to spore formation is the action of a series of sporulation-specific RNA polymerase sigma  factors. Of these, sigma F is the first to become active. Few genes have been identified that are transcribed by RNA polymerase containing sigma F (E-sigma F), and only two genes of known function are exclusively under the control of E-sigma F, spoIIR and spoIIQ. In order to investigate the features of promoters that are recognized by E-sigma F, we studied the effects of randomizing sequences for the -10 and -35 regions of the promoter for spoIIQ. The randomized promoter regions were cloned in front of a promoterless copy of lacZ in a vector designed for insertion by double crossover of single copies of the promoter-lacZ fusions into the amyE region of the B. subtilis chromosome. This system made it possible to test for transcription of lacZ by E-sigma F in vivo. The results indicate a weak sigma F-specific -10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated sigma  factors, as well as to sigma A. There was a rather stronger -35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated sigma  factors. The looseness of the sigma F promoter requirement contrasts with the strict requirement for sigma A-directed promoters of B. subtilis. It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-sigma F in vivo.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140. Phone: (215) 707-7927. Fax: (215) 707-7788. E-mail: piggotp{at}astro.temple.edu.

dagger Present address: Department of Microbiology, University of Colorado Health Science Center, Denver, Colo.

Dagger Present address: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colo.

Journal of Bacteriology, June 2001, p. 3623-3630, Vol. 183, No. 12
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.12.3623-3630.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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