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.

The Zymomonas mobilis genes encoding the glucose facilitator (glf), glucokinase (glk), or fructokinase (frk) were cloned and expressed in a lacIq-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 (Km, 4.1 mM for glucose and 39 mM for fructose; Vmax at 15 degrees C, 75 and 93 nmol min-1 mg-1 [dry weight] for glucose and fructose, respectively). For both substrates, counterflow maxima were observed.     

Expression of the Escherichia coli pmi gene, encoding phosphomannose-isomerase in Zymomonas mobilis, leads to utilization of mannose as a novel growth substrate, which can be used as a selective marker.

Wild-type Zymomonas mobilis can utilize only three substrates (sucrose, glucose, and fructose) as sole carbon sources, which are largely converted into ethanol and carbon dioxide. Here, we show that although D-mannose is not used as a growth substrate, it is taken up via the glucose uniport system (glucose facilitator protein) with a Vmax similar to that of glucose. Moreover, D-mannose was phosphorylated by a side activity of the resident fructokinase to mannose-6-phosphate. Fructokinase was purified to homogeneity from an frk-recombinant Z. mobilis strain showing a specific activity of 205 +/- 25 U of protein mg-1 with fructose (K(m), 0.75 +/- 0.06 mM) and 17 +/- 2 U mg-1 (relative activity, 8.5%) with mannose (K(m), 0.65 +/- 0.08 mM). However, no phosphomannoseisomerase activity could be detected for Z. mobilis, and this appeared to be the reason for the lack of growth on mannose. Therefore, we introduced the Escherichia coli gene pmi (manA) in Z. mobilis under the control of a lacIq-Ptac system on a broad-host-range plasmid (pZY507; Cmr). Subsequently, in pmi-recombinant cells of Z. mobilis, phosphomannoseisomerase was expressed in a range of from 3 U (without isopropyl-beta-D-thiogalactopyranoside [IPTG]) to 20 U mg-1 of protein in crude extracts (after IPTG induction). Recombinant cells of different Z. mobilis strains utilized mannose (4%) as the sole carbon source with a growth rate of 0.07 h-1, provided that they contained fructokinase activity. When the frk gene was additionally expressed from the same vector, fructokinase activities of as much as 9.7 U mg-1 and growth rates of as much as 0.25 h-1 were detected, compared with 0.34 h-1 on fructose for wild-type Z. mobilis. Selection for growth on mannose was used to monitor plasmid transfer of pZY507pmi from E. coli to Z. mobilis strains and could replace the previous selection for antibiotic resistance.     

Kinetics of Sugar Transport and Phosphorylation Influence Glucose and Fructose Cometabolism by Zymomonas mobilis

The competitive inhibition of fructokinase by glucose has been proposed as the mechanism by which Zymomonas mobilis preferentially consumes glucose from mixtures of glucose and fructose and accumulates fructose when growing on sucrose. In this study, incorporation of radioactive fructose into biomass was used as a measure of fructose catabolism. It was determined that the rate of fructose incorporation by Z. mobilis CP4 was somewhat lower in the presence of an equimolar concentration of glucose but that the inhibition of fructokinase by glucose was not nearly as severe in vivo as was predicted from in vitro studies. Interestingly, addition of glucose to a culture of Z. mobilis CP4-M2, a glucokinaseless mutant, resulted in an immediate and nearly complete inhibition of fructose incorporation. Furthermore, addition of nonmetabolizeable glucose analogs had a similar effect on fructose catabolism by the wild-type Z. mobilis CP4, and fructose uptake by Z. mobilis CP4-M2 was shown to be severely inhibited by equimolar amounts of glucose. These results suggest that competition for fructose transport plays an important role in preferential catabolism of glucose from sugar mixtures. Indeed, the apparent K(infm) values for sugar uptake by Z. mobilis CP4 were approximately 200 mM for fructose and 13 mM for glucose. Other experiments supported the conclusion that a single facilitated diffusion transport system, encoded by the glf gene, is solely responsible for the uptake of both glucose and fructose. The results are discussed with regard to the hypothesis that the kinetics of sugar transport and phosphorylation allow the preferential consumption of glucose and accumulation of fructose, making the fructose available for the enzyme glucose-fructose oxidoreductase, which forms sorbitol, an important osmoprotectant for Z. mobilis when growing in the presence of high sugar concentrations.     

Phosphoglycerate kinase gene from Zymomonas mobilis: cloning, sequencing, and localization within the gap operon.

The Zymomonas mobilis gene encoding phosphoglycerate kinase (EC 2.7.3.2), pgk, has been cloned into Escherichia coli and sequenced. It consists of 336 amino acids, including the N-terminal methionine, with a molecular weight of 41,384. This promoterless gene is located 225 base pairs downstream from the gap gene and is part of the gap operon. Previous studies have shown that the specific activities of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase do not change coordinately in Z. mobilis, although the two enzymes appear to be under the control of a common promoter. The translated amino acid sequence for the Z. mobilis phosphoglycerate kinase is less conserved than those of eucaryotic genes. A comparison of known sequences for phosphoglycerate kinase revealed a high degree of conservation of structure with 102 amino acid positions being retained by all. In general, the amino acid positions at the boundaries of beta-sheet and alpha-helical regions and those connecting these regions were more highly conserved than the amino acid positions within regions of secondary structure.     

Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli.

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.     

Hexokinases of Tobacco Leaves: Changes in the Cytosolic and Non-Cytosolic Isozyme Complexes Induced by Tobacco Mosaic Virus Infection

Changes in the cytosotic (soluble) and the non-cytosolic (particulate) isozyme composition of hexokinases and in their properties were studied by ion exchange chromatography on DEAE cellulose after the subcellular fractionation both in the healthy and the tobacco mosaic virus (TMV) infected tobacco leaves. Three main isozyme complexes were obtained: one particulate fraction (the particulate hexokinase phosphorylating both glucose and fructose, EC 2.7.1.1), and two soluble fractions (the soluble hexokinase phosphorylating both the glucose and the fructose, and the soluble fructokinase, which phosphorylates primarily fructose, EC 2.7.1.4). The total fructokinase activities were nearly twice higher than the total glucokinase activities (188.6 % of glucokinase activity in healthy plants and 181.3 % in infected plants). The total particulate glucokinase activity was increased to 120.6 % and the fructokinase to 118.9 % in TMV infected tissue when compared with healthy control. The similar pattern of activity was observed for soluble hexokinase isozymes - the sum of soluble glucokinase activity was increased to 175.4 % and of fructokinase activity to 131.2 % in TMV infected tissue. The isozymes isolated both from the healthy control and TMV-infected leaves had the similar elution profiles, displayed Michaelis-Menten kinetics, showed the identical profiles of pH optima and were Mg²⁺ dependent with the highest enzyme activity at equimolar Mg²⁺ and ATP concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

The existence of two separate constitutive enzymes for glucose and fructose inZymomonas mobilis

Summary Two separate constitutive enzymes responsible for glucose and fructose phosphorylation respectively were identified inZymomonas mobilis grown on sucrose, glucose or fructose. Both enzymes were separated, purified and identified as glucokinase (EC 2.7.1.2) and fructokinase (EC 2.7.1.4). Glucokinase exhibits a molecular weight of 60,000 and fructokinase of 85,000 daltons.     

A Zymomonas mobilis Mutant with Delayed Growth on High Glucose Concentrations

Exponentially growing cells of Zymomonas mobilis normally exhibit a lag period of up to 3 h when transferred from 0.11 M (2%) to 0.55 M (10%) glucose liquid medium. A mutant of Z. mobilis (CU1Rif2), fortuitously isolated, showed more than a 20-h lag period when grown under the same conditions, whereas on 0.55 M glucose solid medium, it failed to grow. The growth of CU1Rif2 on elevated concentrations of other fermentable (0.55 M sucrose or fructose) or nonfermentable (0.11 M glucose plus 0.44 M maltose or xylose) sugars appeared to be normal. Surprisingly, CU1Rif2 cells grew without any delay on 0.55 M glucose on which wild-type cells had been incubated for 3 h and removed at the beginning of their exponential phase. This apparent preconditioning was not observed with medium obtained from wild-type cells grown on 0.11 M glucose and supplemented to 0.55 M after removal of the wild-type cells. Undelayed growth of CU1Rif2 on 0.55 M glucose previously conditioned by the wild type was impaired by heating or protease treatment. It is suggested that in Z. mobilis, a diffusible proteinaceous heat-labile factor, transitionally not present in 0.55 M glucose CU1Rif2 cultures, triggers growth on 0.55 M glucose. Biochemical analysis of glucose uptake and glycolytic enzymes implied that glucose assimilation was not directly involved in the phenomenon. By use of a wild-type Z. mobilis genomic library, a 4.5-kb DNA fragment which complemented in low copy number the glucose-defective phenotype as well as glucokinase and glucose uptake of CU1Rif2 was isolated. This fragment carries a gene cluster consisting of four putative coding regions, encoding 167, 167, 145, and 220 amino acids with typical Z. mobilis codon usage, -35 and -10 promoter elements, and individual Shine-Dalgarno consensus sites. However, strong homologies were not detected in a BLAST2 (EMBL-Heidelberg) computer search with known protein sequences.     

Cloning of the Zymomonas mobilis structural gene encoding alcohol dehydrogenase I (adhA): sequence comparison and expression in Escherichia coli.

Zymomonas mobilis ferments sugars to produce ethanol with two biochemically distinct isoenzymes of alcohol dehydrogenase. The adhA gene encoding alcohol dehydrogenase I has now been sequenced and compared with the adhB gene, which encodes the second isoenzyme. The deduced amino acid sequences for these gene products exhibited no apparent homology. Alcohol dehydrogenase I contained 337 amino acids, with a subunit molecular weight of 36,096. Based on comparisons of primary amino acid sequences, this enzyme belongs to the family of zinc alcohol dehydrogenases which have been described primarily in eucaryotes. Nearly all of the 22 strictly conserved amino acids in this group were also conserved in Z. mobilis alcohol dehydrogenase I. Alcohol dehydrogenase I is an abundant protein, although adhA lacked many of the features previously reported in four other highly expressed genes from Z. mobilis. Codon usage in adhA is not highly biased and includes many codons which were unused by pdc, adhB, gap, and pgk. The ribosomal binding region of adhA lacked the canonical Shine-Dalgarno sequence found in the other highly expressed genes from Z. mobilis. Although these features may facilitate the expression of high enzyme levels, they do not appear to be essential for the expression of Z. mobilis adhA.     

Bifidobacterium longum requires a fructokinase (Frk; ATP:D-fructose 6-phosphotransferase, EC 2.7.1.4) for fructose catabolism

Although the ability of Bifidobacterium spp. to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined. This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in Bifidobacterium longum. The B. longum A10C fructokinase-encoding gene (frk) was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on a Co(2+)-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (Km(fructose) = 0.739 +/- 0.18 mM and Km(ATP) = 0.756 +/- 0.08 mM), is highly specific for D-fructose, and is inhibited by an excess of ATP (>12 mM). It was also found that frk is inducible by fructose and is subject to glucose-mediated repression. Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for D-fructose.     

Simultaneous purification and characterization of glucokinase, fructokinase and glucose-6-phosphate dehydrogenase from Zymomonas mobilis.

The three enzymes glucokinase (EC 2.7.1.2), fructokinase (EC 2.7.1.4) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were isolated in high yield from extracts of Zymomonas mobilis. The principal steps in the isolation procedures involved the use of selected dye-ligand adsorbent columns, with affinity elution of two of the three enzymes. Glucokinase and fructokinase are dimeric proteins (2 X 33000 Da and 2 X 28000 Da respectively) and glucose-6-phosphate dehydrogenase is a tetramer (4 X 52000 Da). Some similarities in the structural and kinetic parameters of the two kinases were noted, but they have absolute specificity for their substrates. Fructokinase is strongly inhibited by glucose; otherwise non-substrate sugars had little effect on any of the three enzymes.     

Factors affecting hexose phosphorylation in Acetobacter xylinum

Fructose was oxidized and converted to cellulose by cells of Acetobacter xylinum grown on fructose or succinate, but not by cells grown on glucose. In resting fructose-grown cells, glucose strongly suppressed fructose utilization. Extracts obtained from fructose- or succinate-grown cells catalyzed the adenosine triphosphate (ATP)-dependent formation of the 6-phosphate esters of glucose and fructose, whereas glucose-grown cell extracts phosphorylated glucose but not fructose. Fructokinase and glucokinase activities were separated and partially purified from cells grown on glucose, fructose, or succinate. Whereas fructokinase phosphorylated fructose only, glucokinase was active towards glucose and less active towards mannose and glucosamine. The optimal pH for the fructokinase was 7.4 and for the glucokinase was 8.5. The K(m) values for the fructokinase were: fructose, 6.2 mm; and ATP, 0.83 mm. The K(m) values for the glucokinase were: glucose, 0.22 mm; and ATP, 4.2 mm. Fructokinase was inhibited by glucose, glucosamine, mannose, and deoxyglucose in a manner competitive with respect to fructose, with K(i) values of 0.1, 0.14, 0.5, and 7.5 mm, respectively. Adenosine diphosphate (ADP) and adenosine monophosphate (AMP) inhibited both kinases noncompetitively with respect to ATP. The K(i) values were: 1.8 mm (ADP) and 2.1 mm (AMP) for fructokinase, and 2.2 mm (ADP) and 9.6 mm (AMP) for glucokinase. Fructose metabolism in A. xylinum appears to be regulated by the synthesis and activity of fructokinase.