Regulatory network for the adaption of Bacillus subtilis to reduced oxygen tension and changes in pH
Zusammenfassung der Projektergebnisse
The anaerobic regulator Fnr of B. subtilis belongs of the Crp/Fnr family of transcription factors. In E. coli Fnr, the [4Fe–4S]2+ cluster is bound by four cysteine residues in the N-terminal domain (C20, C23, C29, and C122) (Green & Guest, 1993, Green et al., 1993). In this project we could show that in contrast to E. coli Fnr, the [4Fe–4S]2+ cluster of B. subtilis Fnr is coordinated by three C-terminally located cysteine residues at position 227, 230, and 235 and one aspartate residue at position 141. In addition, B. subtilis Fnr forms a dimer independent of the presence of oxygen and of the iron–sulfur cluster. These observed significant differences suggest an activation mechanism for B. subtilis Fnr different to that of the E. coli Fnr protein. Under anaerobic conditions, Fnr gets activated by binding of a [4Fe–4S]2+ presumably followed by structural rearrangements that enables DNA binding of the transcription factor via a helix-turn-helix domain. During the project the regulation of alsSD expression and its regulation by AlsR was analyzed in great detail. DNA-binding studies with purified recombinant B. subtilis AlsR at the alsSD promoter in combination with alsSD promoter mutagenesis experiments identified a 19-bp high affinity palindromic binding site (TAAT-N11-ATTA) at position -76 to -58 as RBS and a low affinity site (AT-N11-AT) at position -41 to -27 as ABS. The site directed mutagenesis approach together with promoter deletion, AlsR/ DNA binding studies, DNA-bending studies and in vitro transcription analyses enabled us to postulate a model of AlsR dependent transcriptional regulation. We assume that tetrameric binding of AlsR to the RBS and die ABS in the presence of acetate lead to formation of a higher ordered complex at the alsS promoter that enables binding of the RNA polymerase and subsequent transcriptional activation. Both projects contribute to a better understanding of the regulatory network of B. subtilis for the adaptation to anaerobic growth conditions.
Projektbezogene Publikationen (Auswahl)
- (2011). Aspartate 141 is the fourth ligand of the oxygen-sensing [4Fe-4S]2+ cluster of Bacillus subtilis transcriptional regulator Fnr. J Biol Chem. 286:2017-2021
Gruner, I., Frädrich, C., Böttger, L. H., Trautwein, A. X., Jahn, D. and E. Härtig
- (2012). Regulation of the anaerobic metabolism in Bacillus subtilis. Adv Microb Physiol. 61:195-216
Härtig, E. and D. Jahn
- (2012). The transcription factor AlsR binds and regulates the promoter of the alsSD operon responsible for acetoin formation in Bacillus subtilis. J Bacteriol. 194:1100-1112
Frädrich, C., March, A., Fiege, K., Hartmann, A., Jahn, D. and E. Härtig
(Siehe online unter https://doi.org/10.1128/JB.06425-11) - (2013). Purification, crystallization and preliminary X-ray analysis of the effector domain of AlsR, an LysR-type transcriptional regulator from Bacillus subtilis. Acta Crystallogr Sect F Struct Biol Cryst Commun. 69:581-584
Frädrich, C., Krausze, J., Quade, N., Heinz, D., Jahn, D. and E. Härtig
(Siehe online unter https://doi.org/10.1107/S1744309113010725)
