TY - JOUR
T1 - Assessment of the interaction between the flux-signaling metabolite fructose-1,6-bisphosphate and the bacterial transcription factors CggR and Cra
AU - Bley Folly, Brenda
AU - Ortega, Alvaro D
AU - Hubmann, Georg
AU - Bonsing-Vedelaar, Silke
AU - Wijma, Hein J
AU - van der Meulen, Pieter
AU - Milias-Argeitis, Andreas
AU - Heinemann, Matthias
N1 - © 2018 John Wiley & Sons Ltd.
PY - 2018/8
Y1 - 2018/8
N2 - Bacteria regulate cell physiology in response to extra- and intracellular cues. Recent work showed that metabolic fluxes are reported by specific metabolites, whose concentrations correlate with flux through the respective metabolic pathway. An example of a flux-signaling metabolite is fructose-1,6-bisphosphate (FBP). In turn, FBP was proposed to allosterically regulate master regulators of carbon metabolism, Cra in Escherichia coli and CggR in Bacillus subtilis. However, a number of questions on the FBP-mediated regulation of these transcription factors is still open. Here, using thermal shift assays and microscale thermophoresis we demonstrate that FBP does not bind Cra, even at millimolar physiological concentration, and with electrophoretic mobility shift assays we also did not find FBP-mediated impairment of Cra's affinity for its operator site, while fructose-1-phosphate does. Furthermore, we show for the first time that FBP binds CggR within the millimolar physiological concentration range of the metabolite, and decreases DNA-binding activity of this transcription factor. Molecular docking experiments only identified a single FBP binding site CggR. Our results provide the long thought after clarity with regards to regulation of Cra activity in E. coli and reveals that E. coli and B. subtilis use distinct cellular mechanism to transduce glycolytic flux signals into transcriptional regulation. This article is protected by copyright. All rights reserved.
AB - Bacteria regulate cell physiology in response to extra- and intracellular cues. Recent work showed that metabolic fluxes are reported by specific metabolites, whose concentrations correlate with flux through the respective metabolic pathway. An example of a flux-signaling metabolite is fructose-1,6-bisphosphate (FBP). In turn, FBP was proposed to allosterically regulate master regulators of carbon metabolism, Cra in Escherichia coli and CggR in Bacillus subtilis. However, a number of questions on the FBP-mediated regulation of these transcription factors is still open. Here, using thermal shift assays and microscale thermophoresis we demonstrate that FBP does not bind Cra, even at millimolar physiological concentration, and with electrophoretic mobility shift assays we also did not find FBP-mediated impairment of Cra's affinity for its operator site, while fructose-1-phosphate does. Furthermore, we show for the first time that FBP binds CggR within the millimolar physiological concentration range of the metabolite, and decreases DNA-binding activity of this transcription factor. Molecular docking experiments only identified a single FBP binding site CggR. Our results provide the long thought after clarity with regards to regulation of Cra activity in E. coli and reveals that E. coli and B. subtilis use distinct cellular mechanism to transduce glycolytic flux signals into transcriptional regulation. This article is protected by copyright. All rights reserved.
U2 - 10.1111/mmi.14008
DO - 10.1111/mmi.14008
M3 - Article
C2 - 29923648
SN - 0950-382X
VL - 109
SP - 278
EP - 290
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 3
ER -