Med,a cell-surface localized protein regulating a competence transcription factor gene,comK, in Bacillus subtilis

Med was found as a positive regulator for comK, a master regulator for late competence genes. It was found by Western analysis that the ComK level was decreased in a med mutant. Experiments using an alkaline phosphatase fusion with Med and Western analysis of Med were done because a putative lipo-modification signal is found at the N-terminus of Med. The results obtained are consistent with the localization of Med at the cell surface. An implication of the cell-surface localization of Med is discussed in terms of comK regulation.     

Mutational analysis of the helix-turn-helix region of Bacillus subtilis response regulator DegU, and identification of cis-acting sequences for DegU in the aprE and comK promoters

The DegS-DegU two-component system in Bacillus subtilis regulates exoprotease production and competence development. Phosphorylated and unphosphorylated forms of DegU are required for activation of aprE and comK, respectively. Alanine-scanning mutagenesis of the helix-turn-helix region of DegU and in vivo examination of 27 DegU variants revealed five common mutants that showed severe reduction of gene expression of both aprE and comK because of reduced DNA-binding activity. This observation suggested that the DegU-recognized cis-sequences might not be considerably changed for either promoter. We identified a DegU-recognized inverted repeat in the comK promoter using various mutant comK-lacZ fusions. Inspection of the aprE promoter sequence revealed a tandem repeat consisting of short AT-rich sequences containing a consensus one, 5'-TAAAT-3', which was found in the downstream half of the inverted repeat involved in comK activation. Oligonucleotide-directed replacement of the short AT-rich sequences located in the center of each motif decreased DegU-dependent aprE expression, implying that the repeat is required for the activation of aprE. Based on these results, it was concluded that DegU would function through the inverted repeat in the comK promoter and the tandem repeat in the aprE promoter.     

CodY is required for nutritional repression of Bacillus subtilis genetic competence.

The acquisition of genetic competence by Bacillus subtilis is repressed when the growth medium contains Casamino Acids. This repression was shown to be exerted at the level of expression from the promoters of the competence-regulatory genes srfA and comK and was relieved in strains carrying a null mutation in the codY gene. DNase I footprinting experiments showed that purified CodY binds directly to the srfA and comK promoter regions.