ApoFnr Binds as a Monomer to Promoters Regulating the Expression of Enterotoxin Genes of Bacillus cereus
- 15 June 2008
- journal article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 190 (12), 4242-4251
- https://doi.org/10.1128/jb.00336-08
Abstract
Bacillus cereus Fnr is a member of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of hbl and nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (Fnr His ) fusion protein or an N-terminal fusion protein tagged with the Strep -tag (IBA BioTAGnology) ( Strep Fnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, apoFnr His was mainly monomeric, while apo Strep Fnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apo Strep Fnr was dithiothreitol sensitive, underlining the importance of a disulfide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr , resDE , and plcR and the structural genes hbl and nhe . The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed.Keywords
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