Regulation of Glucose Metabolism in Pseudomonas

Abstract

In Pseudomonas putida, genes for the glucose phosphorylative pathway and the Entner-Doudoroff pathway are organized in two operons; one made up of the zwf, pgl, and eda genes and another consisting of the edd, glk, gltR2, and gltS genes. Divergently with respect to the edd gene is the gap-1 gene. Expression from P zwf , P edd , and P gap is modulated by HexR in response to the availability of glucose in the medium. To study the regulatory process in greater detail we purified HexR and showed that it is a monomer in solution. Electrophoretic mobility shift assays and isothermal titration calorimetry assays were done showing that HexR recognizes the P edd , P zwf , and P gap-1 promoters with affinity in the nanomolar range. DNA footprinting assays identified the binding site between ؉30 and ؉1 at P zwf , between ؉16 and ؉41 at P edd , and between ؊6 and ؉18 at P gap-1 . Based on DNA sequence alignment of the target sites and isothermal titration calorimetry data, two monomers of HexR bind to a pseudopalindrome with a consensus sequence of 5-TTGTN 7-8 ACAA-3. Binding of the Entner-Doudoroff pathway intermediate 2-keto-3-deoxy-6-phosphogluconate to HexR released the repressor from its target operators, whereas other chemicals such as glucose, glucose 6-phosphate, and 6-phosphogluconate did not induce complex dissociation. The phosphorylated effector is likely to be recognized by a sugar isomerase domain located at the C-terminal end of HexR, whereas the helix-turn-helix DNA binding domain of HexR exhibits high similarity to proteins of the RpiR family of regulators.Bacteria of the genus Pseudomonas are ubiquitous inhabitants of soil, water, plant surfaces, and animal tissues. The complete genome sequences of a number of Pseudomonas species and different strains have been deciphered, and their analysis has revealed that Pseudomonas exhibits a limited ability to metabolize sugars; nonetheless, the genomes of all Pseudomonas strains sequenced to date show that they possess the necessary genetic information to metabolize glucose (1-8). In fact, glucose metabolism in Pseudomonas is biochemically rich, as up to three convergent pathways that transform the sugar into 6-phosphogluconate have been described. Subsequently, 6-phosphogluconate is metabolized by Entner-Doudoroff enzymes into central metabolites (9 -13).Glucose metabolism is compartmentalized in Pseudomonas in the sense that once glucose passes the outer membrane through the OprB porin and reaches the periplasm (14 -16), it can be transported to the cytoplasm, or it can be oxidized by the action of the periplasmic glucose dehydrogenase to yield gluconate, which by the action of gluconate dehydrogenase is transformed into 2-ketogluconate. Gluconate and 2-ketogluconate may also be transported to the cytoplasm through a process mediated by the GnuK and KguP transporters, respectively. In the cytoplasm gluconate is directly phosphorylated to 6-phosphogluconate, whereas two reactions mediated by KguK and KguD are needed to convert 2-ketogluconate into 6-phosphoglucona...

Steady-State Hydrogen Peroxide Induces Glycolysis in Staphylococcus aureus and Pseudomonas aeruginosa

“…The transcription of these operons is controlled by the repressor HexR, which directly binds to an inverted repeat (TTGTN 7-8 ACAA) in these two promoters, which is released by the specific binding of the ED pathway intermediate 2-keto-3-deoxy-6-phosphogluconate (KDPG) to HexR (24). The orthologs of these glycolysis ED genes are organized in a similar way in P. aeruginosa, which have not been well characterized (Fig.…”

“…In P. aeruginosa, PPP is closely associated with the Entner-Doudoroff (ED) pathway, which is controlled by the repressor HexR. HexR specifically senses 2-keto-3-deoxy-6-phosphogluconate (KDPG), which results in derepression of several ED operons (24). It has been shown in yeast (Saccharomyces cerevisiae) that upon ROS, oxidative inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) leads to prompt metabolic redirection from glycolysis to PPP, which generates more NADPH (25).…”

HexR Controls Glucose-Responsive Genes and Central Carbon Metabolism in Neisseria meningitidis

“…HexR contains two domains, namely, a helix-turn-helix (HTH) binding domain in the N-terminal region and a sugar isomerase (SIS) domain in the C-terminal region that is predicted to bind phosphosugars. In the gammaproteobacteria P. putida and Shewanella oneidensis, binding of the 2-dehydro-3-deoxyphosphogluconate (KDPG) metabolic intermediate of glycolysis was reported to dissociate HexR from its target DNA (43,44). The NMB1389 (hexR) nucleotide sequence is highly conserved (Ͼ98%) among the available N. meningitidis genome sequences, and it is present in all species of the Neisseria genus, such as Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea, Neisseria flavescens, Neisseria mucosa, Neisseria sicca, and Neisseria subflava.…”

“…1). The NMB1389 gene encodes a protein with 41% amino acid identity and 58% amino acid similarity to the HexR protein of Pseudomonas putida (43). HexR-like transcriptional regulators from the RpiR family are often involved in sugar catabolism regulation in proteobacteria (43,44).…”

“…The NMB1389 gene encodes a protein with 41% amino acid identity and 58% amino acid similarity to the HexR protein of Pseudomonas putida (43). HexR-like transcriptional regulators from the RpiR family are often involved in sugar catabolism regulation in proteobacteria (43,44). HexR contains two domains, namely, a helix-turn-helix (HTH) binding domain in the N-terminal region and a sugar isomerase (SIS) domain in the C-terminal region that is predicted to bind phosphosugars.…”