The competence transcription factor of Bacillus subtilis recognizes short A/T-rich sequences arranged in a unique, flexible pattern along the DNA helix

Abstract

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which leads to the synthesis of the competence transcription factor (CTF). Previous studies suggested that CTF is encoded by comK. ComK is required for the transcription of comK itself, as well as of the late competence genes encoding the DNA uptake machinery and of genes required for homologous recombination. Here, we used purified ComK to study its role in transcription and to determine the DNA recognition sequence for ComK. In vitro transcription from the comG promoter, which depends on ComK in vivo, was observed on the addition of purified ComK together with Bacillus subtilis RNA polymerase, proving that ComK is CTF. To determine the DNA sequences involved in ComK recognition, footprinting analysis was performed with promoter fragments of the CTF-dependent genes: comC, comE, comF, comG, comK, and addAB. The ComK binding sites determined by DNase I protection experiments were unusually long, with average lengths of ∼65 bp, and displayed only weak sequence similarities. Hydroxy-radical footprinting, performed with the addAB promoter, revealed a unique arrangement of four short A/T-rich sequences. Gel retardation experiments indicated that four molecules of ComK bound the addAB promoter and the dyad symmetrical arrangement of the four A/T-rich sequences implied that ComK functions as a tetramer composed of two dimers each recognizing the motif AAAAN 5 TTTT. Comparable A/T-rich sequences were identified in all six DNase I footprints and could be used to predict ComK targets in the B. subtilis genome. On the basis of the variability in distance between the ComK-dimer binding sites, ComK-regulated promoters could be divided into three classes, demonstrating a remarkable flexibility in the binding of ComK.The pattern of hydroxy-radical protections suggested that ComK binds at one face of the DNA helix through the minor groove. This inference was strengthened by the observation that minor groove binding drugs inhibited the binding of ComK.

The pleiotropic response regulator DegU functions as a priming protein in competence development in Bacillus subtilis

“…ComK itself activates the transcription of comK. It has previously been shown that ComK binds upstream of the comK transcription initiation site, where it presumably recruits the RNA polymerase (19). Since DegU also binds to the comK promoter, we tested whether DegU might enhance the binding of ComK.…”

“…Recently, it has been shown that ComK operates as a double dimer, in which each dimer recognizes a dyad symmetrical consensus sequence (AAAAN 5 TTTT), the AT-box (19). In the comK promoter, the distance between the two AT-boxes is 44 bp.…”

“…Gel retardation analyses were carried out as described by Hamoen et al (19). The comK promoter region was isolated by PCR using primers K1 (5Ј-CCG GAA TTC AGA ATC CCC CCA ATG CC-3Ј) and K2 (5Ј-CGG GAT CCC AGT CTG TTT TCT GAC TCA TAT T-3Ј) and B. subtilis 168 chromosomal DNA as template.…”

“…DNase I footprints were obtained as described by Hamoen et al (19). The DNA probes were obtained by PCR amplification using primers K1 and K2 which were endlabeled by using [␥-32 P]ATP.…”

Hereditary stability and variation in evolution and development

“…Transformation competence in bacteria and meiosis in eukaryotes are both differentiated states for generating genetic variation via homologous recombination, and both are bona fide developmental processes. In the well-studied cases of Bacillus subtilis and Sacharomyces cerevisiae the induction of competence (Hamoen et al 1998) and meiosis (Chu et al 1998), respectively, are triggered by nutritional exhaustion and/or cell crowding. To a first approximation, homologous recombination is equally probable anywhere along DNA sequences that are similar (first formulated by JBS Haldane).…”