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A Single, Specific Thymine Mutation in the ComK-Binding Site Severely Decreases Binding and Transcription Activation by the Competence Transcription Factor ComK of Bacillus subtilis

Kim A. Susanna, Aleksandra M. Mironczuk, Wiep Klaas Smits, Leendert W. Hamoen, and Oscar P. Kuipers^*

Department of Genetics, University of Groningen, NL-9751 NN Haren, The Netherlands

Received 21 February 2007/ Accepted 17 April 2007

The competence transcription factor ComK plays a central role in competence development in Bacillus subtilis by activating the transcription of the K regulon. ComK-activated genes are characterized by the presence of a specific sequence to which ComK binds, a K-box, in their upstream DNA region. Each K-box consists of two AT-boxes with the consensus sequence AAAA-(N)₅-TTTT, which are separated by a flexible spacer resulting in either two, three, or four helical turns between the starting nucleotides of the repeating AT-box units. In this study, the effects of potential determinants of ComK regulation in K-boxes were investigated by testing ComK's transcription activation and DNA-binding affinity on altered K-boxes with mutations either in the spacer between the AT-boxes or in the consensus sequence of the AT-boxes. The most striking result demonstrates the importance of the second thymine base in the AT-boxes. Mutation of this T into a guanine resulted in a threefold reduction in transcription activation and DNA binding by ComK. Transcription activation, as well as DNA binding, was almost completely abolished when both AT-boxes contained a T₂-to-G mutation. This result was corroborated by in silico analyses demonstrating that a combination of mutations at the T₂ positions of both AT-boxes is not found among any ComK-activated K-boxes, indicating that at least one consensus T₂ position is required to maintain a functional K-box. The results suggest an important structural role for T₂ in ComK binding, probably by its specific position in the minor groove of the DNA.

Published ahead of print on 27 April 2007.

Present address: Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, P.O. Box 1068, Blindern, 0316 Oslo, Norway.

Present address: Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle, Framlington Place, Newcastle NE2 4HH, United Kingdom.