Assessment of the interaction between the flux‐signaling metabolite fructose‐1,6‐bisphosphate and the bacterial transcription factors CggR and Cra

Abstract

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.

The imbroglio of the physiological Cra effector clarified at last

“…All of them ruled out the idea that FBP could interact with Cra. While this evidence confirms for the E. coli protein what had already been reported for the P. putida regulator, Bley-Folly et al (2018) gives another overpowering screwturn to the case. In a most elegant twist, the same authors could quantify with nuclear magnetic resonance (NMR) the presence of F1P in the preparations of FBP used in their experiments, which turned out to be in the range of 0.002%.…”

“…While the issue of the Cra effectors seems to have been definitely solved by Bley‐Folly et al () the same results open new questions about the role of Cra in regulation of central metabolism. Specifically, the paper makes one ask how could the demonstrated dependence of Cra activity on glycolytic flux take place in vivo .…”

“…Regulation by Cra is mediated solely by F1P and seems to be a general principle in bacteria. The studies of Bley‐Folly et al () and Chavarría et al ( ) definitively ruled out the function of FBP as an effector of Cra. Note that F1P is not part of the core central metabolism and therefore its synthesis has to originate in routes other than the PTS Fru system.…”

“…A cohort of publications (some of the most conspicuous appearing in recent years) build on the notion that Cra is a glycolytic flux sensor because of its interaction with FBP – and metabolic models consider that Cra thereby orchestrates the operation of central carbon metabolism. These papers (Hardiman et al, ; Kotte et al, ; Kochanowski et al, ; ; Wei et al, ; Zhu et al, ; Kim et al, ) may now deserve a revision or reinterpretation in view of Bley‐Folly et al (). It is tempting to speculate that such error propagation could have been avoided if Chavarría et al () had been published in its original form stating that F1P was the one and only Cra effector in P. putida – as well as in E. coli .…”

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

“…Fluorescently-labeled DNA fragments were generated by hybridization of single-stranded forward (labeled with Alexa Fluor-647 at the 5' end) and reverse (unlabeled) oligonucleotides containing the CggR operator sites. The hybridization protocol and respective hybridization efficiency control were performed as described elswhere 32 . Fluorescence was imaged using the Typhoon 9400 (Amersham Biosciences, UK) with the excitation set to 650 nm and the emission set to 655 nm.…”