The spa-box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis.
The subtilin gene cluster (spa) of Bacillus subtilis ATCC 6633 is organized in transcriptional units spaBTC, spaS, spaIFEG and spaRK. Specific binding of the response regulator protein SpaR to spaB, spaS and spaI DNA promoter fragments was shown by means of electromobility shift assays. A repeated pentanucleotide sequence spaced by six nucleotides was identified as SpaR binding motif (spa-box). Saturating mutational analysis of the spa-box by single- and multiple-base-pair substitutions revealed the consensus motif (A/T)TGAT for optimal SpaR binding with the second, third and fifth position being absolutely conservative. Variations in the spacer size between the two pentanucleotide repeats revealed a strong conservation of their relative location. Only DNA with a proximal arrangement of two pentanucleotide repeats showed affinity to SpaR. A 2:1 stoichiometry between SpaR and DNA was obtained by optical biosensor analyses, which corresponds to the binding of two SpaR proteins per spa-box.
Stein
Torsten
T
Institut für Mikrobiologie, Johann Wolfgang Goethe-Universität, Marie-Curie-Str. 9, 60439 Frankfurt am Main, Germany.
Heinzmann
Stefan
S
Kiesau
Peter
P
Himmel
Bettina
B
Entian
Karl-Dieter
KD
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000378:metabolism; D001412:Bacillus subtilis / Q000276:immunology / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001430:Bacteriocins; D001665:Binding Sites; D015374:Biosensing Techniques; D004252:DNA Mutational Analysis / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D055095:Optics and Photonics; D010455:Peptides; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; D004268:DNA-Binding Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D010455:Peptides; C104626:SpaB protein, Bacillus subtilis; C104625:SpaC protein, bacteria; C089313:SpaI protein, Bacillus subtilis; C473791:SpaR protein, Bacillus subtilis; D014157:Transcription Factors; C002836:subtilin