The <i>spa</i>‐box for transcriptional activation of subtilin biosynthesis and immunity in <i>Bacillus subtilis</i>

Abstract

Summary 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 .