A Region in the Bacillus subtilis Transcription Factor Spo0A That Is Important for spoIIG Promoter Activation

Abstract

Spo0A is a DNA binding protein in Bacillus subtilisrequired for the activation of spoIIG and other promoters at the onset of endospore formation. Activation of some of these promoters may involve interaction of Spo0A and the ςAsubunit of RNA polymerase. Previous studies identified two single-amino-acid substitutions in ςA, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo. Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in ςA. We also found that the S231F substitution partially restored use of the spoIIG promoter by the ςA H359R RNA polymerase in vitro. Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for spoIIG promoter activation, I229. This amino acid substitution in Spo0A did not affect repression of abrB transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding. Moreover, the alanine-substituted Spo0A protein activated the spoIIApromoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, ςH-directed transcription. These and other results suggest that the region of Spo0A near position 229 is involved in ςA-dependent promoter activation.

A new mutation in spo0A with intragenic suppressors in the effector domain

“…These di¡erences between transcription from spoIIA and spoIIE promoters should not be due to a di¡erent organisation of DNA OA boxes since the HTH-binding motif and studied mutations are structurally separated. Directed experiments to de¢ne regions required for c A -but not c H -dependent activation have implicated a segment comprising residues 227^240 in the C-terminal domain of Spo0A [8,9]. Our results using mutations which £ank this region suggest that residues 2272 40 are not the only important contact point, and we suggest a role of suv-3 and suv-4 in stabilising the contact of the transcription factor Spo0A with c A .…”

“…Spo0A displays DNA-binding specificity for a 7-bp DNA sequence, 5P-TGTCGAA-3P, the socalled`0A-box' (reviewed in [5]), and, unusually, is able to activate transcription from both c A -and c H -dependent promoters [5^7]. Whilst regions of the C-terminal domain of Spo0A that contact the primary sigma factor c A have been described [8,9], residues which contact c H have yet to be elucidated. The sequence of the e¡ector domain of Spo0A is conserved only amongst Spo0A homologues from other endospore-forming Bacillus and Clostridium species [10].…”

The regulatory network controlling spore formation inClostridium difficile

“…In B. subtilis, sigE transcription is activated by Spo0A and initiated at the SigA-dependent promoter located upstream of spoIIGA (Baldus et al, 1994;Buckner et al, 1998;Hilbert & Piggot, 2004). In turn, SigE is involved in the transcription of numerous genes including spoIIID and gerR encoding two mother cell regulators.…”

Molecular insights into the initiation of sporulation in Gram-positive bacteria: new technologies for an old phenomenon

“…The structure comprises six a-helices, a A -a F , two of which (a C -a D ) form a classical helix-turn-helix (HTH) DNA-binding motif. The region of Spo0A in the immediate vicinity of a E has been shown to be important in transcription activation from r A -dependent promoters [40][41][42][43], and this part of the structure forms a flexible section in close proximity to the recognition helix of the HTH. It is not yet clear whether the same region, or that defined by the extreme C-terminus of Spo0A, is required for r H -dependent transcription activation [44].…”