Fructose 1-Phosphate Is the Preferred Effector of the Metabolic Regulator Cra of Pseudomonas putida

Abstract

The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of Gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5-TTAAACGTTTCA-3 (K D ‫؍‬ 26.3 ؎ 3.1 nM) and that F1P binds the protein with an apparent stoichiometry of 1.06 ؎ 0.06 molecules per Cra monomer and a K D of 209 ؎ 20 nM. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida.The central metabolism of bacteria is controlled through the interplay of global and specific regulators, the outcome of which depends on the nature of the carbon and energy sources and the particular culture conditions (1). The catabolite repressor/activator (Cra) protein (also known as FruR) is a pleiotropic regulatory protein that plays a key role in the control of carbon flow in Escherichia coli and Salmonella typhimurium (2). This factor was first identified as a repressor that inhibited expression of the fructose operon of these two bacteria when the sugar was not available, thereby the earlier name FruR (3). Later work, however, revealed that the same protein represses genes for many other enzymes of the central metabolism (pfkA, pykA, pykF, acnB, edd, eda, mtlADR, and gapB; and activates others (ppsA, fbp, pckA, acnA, icd, aceA, and aceB; Refs. 2 and 7-10), suggesting a dual character of Cra as both a transcriptional repressor and an activator.From a structural point of view, the Cra protein has been classified as a member of the GalR-LacI superfamily of DNAbinding transcriptional regulators (11,12). In E. coli, the Cra monomer is organized in two functional domains. The N-terminal helix-turn-helix module accounts for the binding of the protein to the cognate DNA operator sequence, whereas the C-terminal portion mediates the interactions between subunits and triggers changes in the protein upon effector binding (12).Earlier studies of the Cra proteins of E. coli and S. typhimurium suggested that the regulator is a tetramer that recognizes an imperfect palindromic DNA sequence to which it bin...

A Metabolic Widget Adjusts the Phosphoenolpyruvate-Dependent Fructose Influx in Pseudomonas putida

“…7B) with respect to the wild-type strain. In the wild-type strain, the derepression of the genes encoding the PTS Fru involves the buildup of enough F1P and the binding of this metabolite to Cra P. putida , thereby bringing about a conformational change in the protein to break free from the target DNA regions (22); i.e., an overall transcriptional adaptation process is needed to start growing on Fru. The shorter lag phase of the Δ cra strain is to be expected since such transcriptional adaptation (and the ensuing derepression of the genes encoding PTS Fru ) is not relevant in this genetic background.…”

“…1A). However, the corresponding Cra P. putida protein does not respond to FBP at all but solely to fructose-1-P (F1P) (21, 22), a metabolite that can be formed only upon the action of PTS Fru on Fru. In sum, when the Fru transport systems of E. coli and P. putida are compared, the same basic constituents are found: fruB, fruA (along with a third gene needed for F1P phosphorylation, fruK ), the Cra repressor, and the phosphorylated forms of Fru (i.e., F1P and FBP) as the effectors.…”

“…In enterobacteria, Cra is a transcriptional regulator that controls the expression of a large number of genes in central carbon metabolism (CCM) according to the distribution of glycolytic fluxes (24–26). In contrast, Cra P. putida strongly binds an operator at the P fruB promoter in a fashion exclusively dependent on the intracellular F1P concentration (22). The lack of other Cra P. putida targets in the chromosome of strain KT2440 is somewhat intriguing considering the plethora of CCM genes regulated by the orthologue in enterobacteria.…”

Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra)

“…EMSA was performed as previously described28. The 18-bp DNA fragment used for EMSA was prepared by heating a 50 nM mixture of complementary oligonucleotides containing the presumed Cra binding site of the aceBAK region in 1 mL of TE buffer at 95 °C for 10 min.…”

ThefruRBAOperon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood

“…1). The physiological inducer of FruR derepression is fructose-1-phosphate (F1P), and only 1 mM F1P is sufficient to disrupt FruR binding to its operator in Pseudomonas putida (55,56). Fructose-1-phosphate is also the inducer of the fruRBA operon in other Gram-positive species, such as L. lactis (18).…”