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.
Shimane
Kana
K
Department of Marine Science and Technology, Tokai University, 3-20-1 Shimizu-Orido, Shizuoka 424-8610, Japan.
Ogura
Mitsuo
M
D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D015153:Blotting, Western; D002701:Chloramphenicol / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D004917:Erythromycin / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010641:Phenotype; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics*
C088074:AprE protein, Bacteria; D001426:Bacterial Proteins; D017931:DNA Primers; C066496:DegS protein, Bacteria; C066497:DegU protein, Bacteria; D026901:Membrane Transport Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C087449:comK protein, Bacillus subtilis; D004917:Erythromycin; D002701:Chloramphenicol; D004247:DNA; D000409:Alanine