Functional expression of the glucose transporter of Zymomonas mobilis leads to restoration of glucose and fructose uptake in Escherichia coli mutants and provides evidence for its facilitator action

Abstract

The Zymomonas mobilis genes encoding the glucose facilitator (glf), glucokinase (glk), or fructokinase (frk) were cloned and expressed in a lacI q -Ptac system using Escherichia coli K-12 mutants deficient in uptake and phosphorylation of glucose and fructose. Growth on glucose or fructose was restored when the respective genes (glf-glk or glf-frk) were expressed. In E. coli glf ؉ strains, both glucose and fructose were taken up via facilitated diffusion (K m , 4.1 mM for glucose and 39 mM for fructose; V max at 15؇C, 75 and 93 nmol min ؊1 mg ؊1 [dry weight] for glucose and fructose, respectively). For both substrates, counterflow maxima were observed.Glucose can be transported in bacteria by carrier systems which belong either to the phosphoenolpyruvate-dependent phosphotransferase system (PTS) type, to proton-or cationlinked permeases, or to ATP-binding cassette (ABC)-type carriers (4, 17). A rare case is the facilitator-type transport (uniport) of glucose in bacteria, exemplified by the system of the gram-negative, strictly fermentative bacterium Zymomonas mobilis. This type of sugar uptake does not use metabolic energy in the form of a proton potential or phosphoenolpyruvate (10, 17). Biochemical evidence for facilitated diffusion of glucose in this organism came from earlier studies (10, 26) and from in vivo nuclear magnetic resonance measurements using 13 C-labeled glucose and xylose as substrates (22). Mainly on the basis of competition studies, it was stated that the glucose facilitator (GLF) of Z. mobilis is a low-affinity, high-velocity carrier which in addition to glucose also transports fructose, xylose, or nonmetabolizable glucose analogs, such as 2-deoxyglucose (10,22,26). The sequence of a presumptive glf gene from Z. mobilis was reported (6), and on the basis of sequence comparison, this open reading frame was found to be a member of a superfamily of sugar transporters from both prokaryotes and eukaryotes (for a recent review, see reference 3). Recently, it was shown that the glf plus glk genes of Z. mobilis complemented an Escherichia coli mutant strain, which was deficient in glucokinase and in glucose uptake, to growth on glucose (23). However, detailed glucose uptake studies using this heterologous system have not yet been reported, nor was the substrate range of this carrier studied further.Plasmid constructions. DNA preparation, cloning, restriction analysis, and transformation were done according to established methods (20). Chromosomal DNA from Z. mobilis ZM6 (27) was prepared according to the lysozyme freeze-thaw method (12). The Z. mobilis gene glf from strain CP4 (15) had been fortuitously cloned in our laboratory as a 2.3-kb HindIII DNA fragment (pZY600) and shown to confer Glf activity to E. coli Glc Ϫ strains (18). DNA sequencing established the identity with the glf sequence reported earlier (6). The glf gene was amplified by PCR (16) using plasmid pZY600 as a template, and glk (6) and frk (31) genes were amplified from chromosomal DNA of Z. mobilis ZM6. The PCR primers for glf wer...

In Silico and Transcriptional Analysis of Carbohydrate Uptake Systems of Streptomyces coelicolor A3(2)

“…In S. coelicolor, fructose, N-acetylglucosamine, and possibly two further carbohydrates are transported by the phosphotransferase system (PTS) (29,30,33,54). A homologous PTS specific for N-acetylglucosamine was also found in S. olivaceoviridis (61).In this report, we provide a compilation of carbohydrate uptake systems present in S. coelicolor. In addition to the few already known transport systems, we identified new potential ones by similarity searches of the genome DNA-protein data bank with well-characterized prokaryotic and eukaryotic carbohydrate permeases.…”

“…In S. coelicolor, fructose, N-acetylglucosamine, and possibly two further carbohydrates are transported by the phosphotransferase system (PTS) (29,30,33,54). A homologous PTS specific for N-acetylglucosamine was also found in S. olivaceoviridis (61).…”

Structure and gene-polypeptide relationships of the region encoding glycerol diffusion facilitator (glpF) and glycerol kinase (glpk) of Pseudomonas aeruginosa

“…Weissenborn & Larson, 1992) and the glucose facilitator of Zymomonas mobilis (Glf) (Parker et al, 1995;Weisser et al, 1995). Although glycerol, like many other small solutes, can enter the cytoplasm of E. coli via passive diffusion, effective glycerol uptake was shown to be protein-mediated (Sanno et al, 1968) and dependent on phosphorylation to sn-glycerol 3-phosphate (G3P) (Voegele et al, 1993).…”

Bifidobacterium longum Requires a Fructokinase (Frk; ATP: d -Fructose 6-Phosphotransferase, EC 2.7.1.4) for Fructose Catabolism

“…The phosphotransferase system involves two classes of proteins, sugar-specific (EII) and general (EI and HPr) enzymes, which supply the cell with fructose-1-P (16,32,35), whereas fructokinases (Frk or ScrK; ATP:␤-D-fructose 6-phosphotransferases) catalyze the transfer of ␥-phosphate from ATP to an intracellular molecule of fructose, leading to fructose-6-P (F6P) (3,39,40,66). For the majority of bacteria, genes encoding fructokinases are either isolated on the chromosome (frk) (17,64,65,66) or members of a sucrose utilization gene cluster (scrK, sacK, or cscK) (3,5,6,28,38,53).…”