Metabolism of Carbohydrate Derivatives by Pseudomonas

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1 JOURNAL OF BACTERIOLOGY, May 1979, p /79/ /07$02.00/0 Vol. 138, No. 2 Metabolism of Carbohydrate Derivatives by Pseudomonas acidovorans MICHEL H. WETTERMARK,t JULIE R. TAYLOR,4 MICHAEL L. ROGERS, AND H. E. HEATH* Department ofmicrobiology, The University ofalabama, University, Alabama Received for publication 6 March 1979 Wild-type Pseudomonas acidovorans strain Al was unable to grow on glycerol or glucose as sole source of carbon and energy although it grew well on gluconate. Spontaneous glycerol-positive mutants, which apparently had become permeable to glycerol, were readily isolated, but glucose-positive mutants did not occur. P. acidovorans lacked glucose dehydrogenase and glucokinase, which were sufficient to account for its inability to grow on glucose. Gluconate was degraded exclusively via a noncoordinately induced Entner-Doudoroff pathway. Phosphogluconate dehydrogenase was undetectable. In contrast to P. aeruginosa, P. acidovorans possessed a single glyceraldehyde-phosphate dehydrogenase activity, which was NAD+ specific and constitutive, and an inducible pyruvate kinase. Moreover, growth of glycerol-positive strain K2 on glycerol did not induce any of the enzymes related to metabolism of hexosephosphate derivatives as occurs in fluorescent pseudomonads. Pseudomonas acidovorans is a metabolically versatile nonfluorescent aerobic pseudomonad. Although this species is able to grow on gluconate as the sole source of carbon and energy, it does not grow on glucose or 2-ketogluconate, and only 5 of 15 strains tested are able to grow on glycerol (22). Among the metabolically versatile pseudomonads, these nutritional disabilities are unique to P. acidovorans and the closely related (14, 15) species P. testosteroni (22). Catabolism of glucose and related compounds by P. aeruginosa and other members of the fluorescent group is complex (Fig. 1). Furthermore, growth of P. aeruginosa on glycerol induces most of the enzymes involved in degradation of glucose (8-10, 19). This occurs because growth of P. aeruginosa on glycerol requires formation of a catabolic NAD+-linked glyceraldehyde-phosphate dehydrogenase, which is induced by a hexosephosphate derivative that inhibits growth if not degraded (8). Because P. acidovorans is unable to grow on glucose or 2-ketogluconate, it appears to offer a simplified system in which to analyze metabolism of carbohydrate derivatives. This report is a general survey of the metabolic routes involved in utilization of gluconate. The results clarified the biochemical basis for the inability of wildtype strain Al to grow on glycerol or glucose. Characterization of a glycerol-positive mutant t Present address: College of Medicine, University of South Alabama, Mobile, AL t Present address: Akwell Industries, Dothan, AL also revealed that, unlike P. aeruginosa, growth of P. acidovorans on glycerol did not induce the enzymes for metabolism of hexosephosphate derivatives. MATERIALS AND METHODS Bacterial strains. Wild-type P. acidovorans Al (ATCC type strain 15668, strain 14 of Stanier et al. [22]) was obtained from G. D. Hegeman, Indiana University. Strain K2 was selected for resistance to streptomycin (1.0 mg/ml) from strain S1, which was a spontaneous glycerol-positive mutant of strain Al. P. aeruginosa 1 (PAO, ATCC 15692, strain 131 of Stanier et al. [22]) was provided by T. W. Feary, Louisiana State University Medical Center. Escherichia coli B (ATCC 11303) was from the collection of J. W. Drake, National Institute of Environmental Health Sciences. Stock cultures were stored at -15 C in 0.7% peptone (Difco) containing 30% (vol/vol) glycerol. Growth media. A complex medium containing 1.0% tryptone (Difco), 0.5% yeast extract (Difco), and 0.5% NaCl was used for routine growth of cells. For preparation of crude extracts, E. coli was grown in nutrient broth (Difco) supplemented with 0.5% glucose. Liquid minimal medium (K9) contained 40 mm K2HPO4, 20 mm KH2PO4, 50 mm NaCl, 20 mm NH4Cl, 1.0 mm MgSO4, and 0.01 mm FeSO4. The final concentrations of substrates were 10 mm glucose, 20 mm glycerol, 10 mm mannitol, 10 mm sodium gluconate, 20 mm sodium D,L-glycerol 3-phosphate, or 20 mm sodium lactate. Solid minimal medium was an appropriately supplemented phosphate-ammoniumsalts mixture described by Chakrabarty et al. (3). Growth of cultures. Cultures of P. acidovorans were grown at 30 C; cultures of P. aeruginosa and E. coli were grown at 37 C. Liquid cultures, not exceeding

2 VOL. 138, 1979 CARBOHYDRATE METABOLISM IN P. ACIDOVORANS 419 Glco0 G30 o,o e 2 Glucose Glucontec Pentose-P /o 0 \9 \ / I Lactate \ \ ~~/ \ /25 /2 Pyruvate de yde-3-p 0 \6 occ 40o-yP Xf\ e4 3-P-Glyceroyl-P P-Eno/pyruvate sn-glycerol-3-p Glycerol 3-P-Glycerate 2-P-Glycerate FIG. 1. Metabolism of carbohydrate derivatives by Pseudomonas. All reactions shown occur in P. aeruginosa. Solid arrows represent enzymatic activities demonstrated in P. acidovorans. Broken arrows indicate reactions presumed to occur. Arrows broken by 0 designate undetectable enzymatic activities. Arrows broken by x indicate reactions presumed to be absent from P. acidovorans. Numbers refer to the following enzymes: 1, glucokinase; 2, glucose-6-phosphate dehydrogenase; 3, glucose dehydrogenase; 4, gluconokinase; 5, gluconate 2-dehydrogenase; 6, ketogluconokinase; 7, 2-keto-6-phosphogluconate reductase; 8, phosphogluconate dehydrogenase; 9, reversible nonoxidative enzymes of the hexose monophosphate pathway; 10, phosphogluconate dehydratase; 11, PKDG aldolase; 12a, glyceraldehyde-phosphate dehydrogenase; 12b, glyceraldehydephosphate dehydrogenase (NADP+) (phosphorylating); 13, phosphoglycerate kinase; 14, phosphoglyceromutase; 15, enolase; 16, pyruvate kinase; 17, triosephosphate isomerase; 18, fructose-bisphosphate aldolase; 19, fructose-bisphosphatase; 20, glucosephosphate isomerase; 21, mannitol dehydrogenase; 22, fructokinase; 23, glycerol kinase; 24, glycerol-3-phosphate dehydrogenase; 25, lactate dehydrogenase. The Entner-Doudoroff pathway consists of enzymes 4, 10, and 11. The anabolic portion of the EMPpathway is composed of enzymes 18, 19, and 20. The triosephosphate pathway (lowerportion of the EMP pathway) consists of enzymes 12a, 13, 14, 15, and % of the nominal volume of the container, were aerated on a rotary shaker at 300 rpm. Growth was measured as absorbance at 540 nm. Enzyme assays. Cultures were grown to late exponential phase (absorbance at 540 nm of approximately 1.4) in the indicated medium. The cells were washed at 0 C with saline, concentrated approximately 30-fold in 50 mm Tris-hydrochloride (ph 7.5) containing 10 mm 2-mercaptoethanol, and ruptured by intermittent 15-s exposures to approximately 300 W of sonic power. Unbroken cells and debris were removed by centrifugation (26,000 x g for 10 min) at 00C. Enzyme assays were performed at ambient temperature (approximately 22 C); the buffers contained 10 mm 2-mercaptoethanol unless otherwise indicated. A unit of enzyme activity (U) was defined as the amount required to convert 1.0 manol of substrate(s) to product(s) per min. The following millimolar extinction coefficients were used to calculate enzyme activities: formazan product of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-2h-tetrazolium bromide reduction at 550 nm, 8.1 (21); NAD(P)H at 340 nm, 6.2 (13); and phosphoenolpyruvate at 230 am, 3.0 (29). Protein was determined by the method of Sutherland et al. (23). Specific activities were calculated in milliunits per

3 420 WETTERMARK ET AL. milligram of protein. The following enzymes were assayed according to the referenced procedure: glycerol kinase (11); glycerol-3-phosphate dehydrogenase (11); triosephosphate isomerase (2); glucokinase (24); glucose-6-phosphate dehydrogenase (24); glucose dehydrogenase (8); gluconate 2-dehydrogenase (8); gluconokinase (8); phosphogluconate dehydrogenase (24); fructose-bisphosphatase (7, 24); glucosephosphate isomerase (7, 24); glyceraldehyde-phosphate dehydrogenase (24); phosphoglycerate kinase (2); enolase (28); and pyruvate kinase (25). Phosphogluconate dehydratase was assayed as conversion of 6-phosphogluconate to pyruvate by oxidation of NADH in the presence of excess phospho-2- keto-3-deoxy-gluconate (PKDG) aldolase and lactate dehydrogenase. The reaction mixture contained 50 mm Tris-hydrochloride (ph 7.5), 2.0 mm MgCl2, 0.2 mm NADH, approximately 0.15 U of PKDG aldolase, 1 U of lactate dehydrogenase, and 2.0 mm 6-phosphogluconate. The PKDG aldolase was provided as 0.1 ml of an extract of E. coli cells grown in nutrient broth supplemented with 0.5% glucose. Such extracts lacked detectable (<1.0 mu) phosphogluconate dehydratase activity (8). Measurement of PKDG aldolase was similar except that exogenous PKDG aldolase was omitted and PKDG was generated in situ by the excess endogenous phosphogluconate dehydratase. Fructose-bisphosphate aldolase was determined as fructose-1,6-bisphosphate-dependent oxidation of NADH in the presence of excess triosephosphate isomerase and glycerol-3-phosphate dehydrogenase. The reaction mixture contained 50 mm Tris-hydrochloride (ph 8.5) without 2-mercaptoethanol, 100 mm KCI, 0.7 mm CoC12, 0.1 mm cysteine-hydrochloride, 0.2 mm NADH, 1 U of triosephosphate isomerase, 1 U of glycerol-3-phosphate dehydrogenase, and 1.0 mm fructose 1,6-bisphosphate. The extract was dialyzed exhaustively against 50 mm Tris-hydrochloride (ph 7.5) to remove 2-mercaptoethanol. Two moles of NADH was assumed to be oxidized per mole of fructose 1,6-bisphosphate cleaved because the exces triosephosphate isomerase would convert glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. The same consideration applied to measurement of 6- phosphofructokinase as fructose-6-phosphate-dependent oxidation of NADH in the presence of excess fructose-bisphosphate aldolase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase. This reaction mixture contained 50 mm Tris-hydrochloride (ph 7.5), 10 mm MgCI2, 2.0 mm ATP, 0.2 mm NADH, 1 U of fructose-bisphosphate aldolase, 1 U of triosephosphate isomerase, 1 U of glycerol-3-phosphate dehydrogenase, and 2.0 mm fructose 6-phosphate. Phosphoglyceromutase was assayed as 3-phosphoglycerate-dependent formation of phosphoenolpyruvate in the presence of 2,3-bisphosphoglycerate and excess enolase. The reaction mixture contained 50 mm Tris-hydrochloride (ph 7.5), 5.0 mm MgCI2, 1.0 mm 2,3-bisphosphoglycerate, 1 U of enolase, and 2.0 mm 3-phosphoglycerate. Chemicals and enzymes. D,L-Glyceraldehyde 3- phosphoric acid was prepared from the barium salt of the diethylacetal derivative by ion exchange with Dowex 50 and subsequent hydrolysis for 3 min at 1000C. Commercial enzymes were the highest quality available from Sigma Chemical Co. All other chemicals were reagent grade or the highest purity available. RESULTS Glycerol-catabolic defect of wild-type strain Al. P. acidovorans Al was unable to grow on glycerol-minimal medium. However, the wild type grew, although poorly, on glycerol 3-phosphate (0.037 doublings per h). Moreover, spontaneous glycerol-positive mutants occurred at a frequency of approximately 5 x 10-7 in cultures of strain Al. Strain K2 was one such derivative. The mutation conferring growth on glycerol (0.24 doublings per h) did not affect growth on glycerol 3-phosphate. Strain K2 possessed inducible glycerol kinase and constitutive glycerol-3-phosphate dehydrogenase and triosephosphate isomerase for conversion of glycerol to glyceraldehyde 3-phosphate (Table 1). Growth of strain K2 or Al on glycerol 3-phosphate also induced glycerol kinase; furthermore, strain Al possessed glycerol-3-phosphate dehydrogenase. Therefore, the wild type is presumably defective for transport of glycerol. Glucose-catabolic defect of strain K2. Strains A1 and K2 were unable to grow on glucose-minimal medium, and numerous attempts to select glucose-positive mutants have failed. As shown in Table 2, strain K2 did not possess all the necessary enzymes for degradation of glucose via either route known in Pseudomonas (Fig. 1). Glucokinase was undetectable under all conditions tested. However, strain K2 did possess glucose-6-phosphate dehydrogenase, which was induced only by growth on mannitol. This activity was nonspecific with respect to pyridine nucleotide; NAD+ gave essentially the TABLE 1. J. BACTERIOL. Enzymes ofglycerol catabolism same results as NADP+ (Table 2). In addition, strain K2 lacked detectable glucose dehydrogen- Glycerol- Strain Substrate Glycerol 3-phos- Triosekinam phate de- phosphate hydro- isomerase genase K2 Lactate ,120 K2 Glycerol ,000 K2 Glycerol NAa phosphate Al Glycerol NA phosphate a NA, Not assayed.

4 VOL. 138, 1979 TABLE 2. Glucose-catabolic defects ofp. acidovorans strain K2 ase under all conditions tested (Table 2). The standard assay readily detected this membraneassociated (12) activity in similarly prepared extracts of gluconate-grown cells of P. aeruginosa. Dissimilation of gluconate by strain K2. Gluconate was degraded exclusively via an inducible Entner-Doudoroff pathway (Table 3). Neither strain Al nor strain K2 grew on 2-ketogluconate, and, as expected, strain K2 lacked gluconate 2-dehydrogenase under all conditions tested (Table 3). Phosphogluconate dehydrogenase was not detected with either pyridine nucleotide or with 3-(4,5-dimethylthiazol-2-yl)- Glucose-6-phos- Substrate Glucoki_ phate dehydro- Glucose genase nase dehydrogenase NAD+ NADP+ Lactate oa ob 0.9 o0 Gluconate Mannitol Glycerol 'Undetectable activity (<1.5 mu); undetectable in cells grown on mannitol in the presence of glucose; gluconate-grown P. aeruginosa had 69.5 mu/mg of protein. b C Undetectable activity (<1.5 mu). Undetectable activity (<1.0 mu) in the standard assay or with either pyridine nucleotide (<1.5 mu); undetectable in cells grown on lactate in the presence of glucose; similarly prepared extracts of gluconategrown P. aeruginosa had 42.6 mu/mg of protein. TABLE 3. Enzymes related to gluconate degradation by strain K2 Glu- Phos- Pho Phos 2-dehyGucn conate conate PD drogeen drge- okinase dehy- dhda aldolase drogen- uethy8draase dge- tase Substrate conate Glucon-pogl phoglu- ase Lactate oa 1.5 0b Gluconate Mannitol Glycerol " Undetectable activity (<1.0 mu) in the standard assay or with either pyridine nucleotide (<1.5 mu); similarly prepared extracts of gluconate-grown P. aeruginosa had 127 mu/mg of protein. bundetectable activity with either pyridine nucleotide (<1.5 mu) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide in the presence of phenazine methosulfate (<1.0 mu); E. coli grown in nutrient broth supplemented with 0.5% glucose had 175 mu/mg of protein (NADP+ linked). CARBOHYDRATE METABOLISM IN P. ACIDOVORANS 421 2,5-diphenyl-2H-tetrazolium bromide in the presence of phenazine methosulfate (Table 3). Growth of strain K2 on gluconate induced gluconokinase, phosphogluconate dehydratase, and PKDG aldolase. However, growth on mannitol induced only gluconokinase, and growth on glycerol did not induce any of these enzymes. Role of the EMP pathway in P. acidovorans. Because P. acidovorans strain K2 was unable to form pentosephosphate directly from 6-phosphogluconate via the oxidative hexose monophosphate pathway, the function of the Embden-Meyerhof-Parnas (EMP) pathway was examined. As expected, strain K2, like other aerobic pseudomonads (4), lacked a catabolic EMP pathway due to the absence of 6-phosphofructokinase (Table 4). However, the necessary enzymes for conversion of triosephosphate to hexosephosphate were consitutive. This presumably permits formation of pentosephosphate and related essential metabolites from triosephosphate and hexosephosphate via the reversible nonoxidative reactions of the hexose monophosphate pathway (5; Fig. 1). The reason for lower levels of fructose-bisphosphate aldolase in cells grown on gluconate, mannitol, or glycerol compared to cells grown on lactate is not yet clear, but it may reflect greater availability of triosephosphate from catabolism of gluconate and glycerol than from metabolism of lactate for formation of hexosephosphate, which also is formed readily from mannitol (cf. Fig. 1). The possible significance of relatively low fructosebisphosphatase and high glucosephosphate isomerase levels in mannitol-grown cells will be considered later. Enzymes of the triosephosphate pathway. Strain K2 possessed a constitutive amphibolic NAD+-linked glyceraldehyde-phosphate dehydrogenase activity (Table 5); no NADP+linked activity was detected. Phosphoglycerate kinase, phosphoglyceromutase, and enolase TABLE 4. Enzymes of the EMP pathway in strain K2 Substrate 6-Phos- Fructose- Fructose- Glucosephofruc- bisphos- bisphos- phosphate tokinase phatedala phatase isomerase Lactate oa Gluconate Mannitol Glycerol NAb e Undetectable activity (<1.0 mu); E. coli grown in nutrient broth supplemented with 0.5% glucose had 57.5 mu/mg of protein. b NA, Not assayed.

5 422 WETTERMARK ET AL. TABLE 5. Enzymes of the triosephosphate pathway in strain K2 Glyceral- Substrate dehyde- Phos- Phosphos- phogly- Pyru- phogly- Eno- vate kiphate cerate ceromu- lase na dehydro- kinase tase genasea Lactate Gluconate Glycerol a Undetectable NADP'-linked activity (<1.5 mu). were also constitutive and therefore presumably amphibolic. However, pyruvate kinase was inducible (Table 5). Growth on mannitol also induced pyruvate kinase (264 mu per mg of protein). DISCUSSION Most strains of P. acidovorans are unable to grow on glycerol (22). This study indicated that wild-type strain Al is defective for transport of glycerol because cells grown on glycerol 3-phosphate had all the enzymes needed for glycerol catabolism. Moreover, the data suggested that glycerol 3-phosphate is the inducer for glycerol kinase. Stanier et al. (22) suggested that the inability of P. acidovorans to grow on glucose may be due to impermneability. However, the catabolic defects related to this nutritional disability were more extensive than this. Transport of glucose may not be required for degradation because glucose dehydrogenase apparently acts extracellularly in P. aeruginosa (12, 19). However, P. acidovorans did not have this enzyme. Furthermore, even if the cells were permeable to glucose, they lacked glucokinase. These facts are therefore sufficient to account for the inability of P. acidovorans to grow on glucose regardless of perneability. P. acidovorans degraded gluconate solely via the Entner-Doudoroff pathway. A similar conclusion was reached for the related species (15), P. saccharophila, which also lacks phosphogluconate dehydrogenase (5). It has been suggested that this activity observed in fluorescent pseudomonads is actually an artifact due to coupling of phosphogluconate dehydratase, PKDG aldolase, and glyceraldehyde-phosphate dehydrogenase (1). However, several lines of evidence against this proposed artifactual coupling in crude extracts have been presented recently (8). The present study provided further evidence that such a coupling does not contribute to phosphogluconate dehydrogenase activity be- J. BACTERIOL. cause crude extracts of P. acidovorans had the three coupling enzymes and yet lacked the predicted NAD+-linked activity. Induction of the enzymes of the Entner-Doudoroff pathway was noncoordinate. Growth on gluconate induced gluconokinase, phosphogluconate dehydratase, and PKDG aldolase, but growth on mannitol only induced gluconokinase. These results suggested that 6-phosphogluconate or possibly PKDG is the inducer for gluconokinase for the following reason. Mannitolgrown cells of strain K2 possess mannitol dehydrogenase and fructokinase (J. R. Taylor, unpublished data) in addition to elevated levels of glucosephosphate isomerase and glucose-6- phosphate dehydrogenase. As intermediates common to mannitol and gluconate metabolism, 6-phosphogluconate or possibly PKDG is therefore likely to be the inducer for gluconokinase. Conversely, gluconate appears to be the inducer for phosphogluconate dehydratase and PKDG aldolase as in P. fluorescens (6). Compared to P. aeruginosa, P. acidovorans exhibited two regulatory differences that result in economy of protein synthesis. Growth of P. aeruginosa on glycerol induces most of the enzymes involved in glucose catabolism (8-10, 19). Similar, though less extensive, observations have been reported for P. putida (27) and P. fluorescens (18). P. aeruginosa has a constitutive amphibolic NADP+-linked glyceraldehyde-phosphate dehydrogenase, but degradation of glyceraldehyde 3-phosphate apparently requires induction of an additional catabolic NAD+-linked activity by a six-carbon metabolite of glucose (8). Conversion of glycerol-derived triosephosphate to hexosephosphate derivatives for induction of this activity also causes induction of the glucose-catabolic enzymes. In contrast, P. acidovorans had only an NAD+-linked glyceraldehyde-phosphate dehydrogenase, which was constitutive and therefore presumably amphibolic. Formation of the enzymes involved in metabolism of hexosephosphate derivatives would be wasteful here. Consistent with this, growth of P. acidovorans on glycerol did not induce any of these enzymes. Pyruvate kinase, which presumably is strictly catabolic, provided another example of improved efficiency because this enzyme is constitutive in P. aeruginosa (8, 24) but inducible in P. acidovorans. As an alternative to induction, repression by pyruvate or its metabolites seemed Unlikely. Essentially the same level of pyruvate kinase was attained by growth on gluconate, which produces glyceraldehyde 3-phosphate and pyruvate directly, or by growth on glycerol, which produces pyruvate indirectly from glyceraldehyde 3-phosphate. This enzyme is there-

6 VOL. 138, 1979 fore presumably induced by phosphoenolpyruvate or a precursor thereof. This study raised questions regarding the pathway for degradation of mannitol in P. acidovorans. Evidence that P. aeruginosa degrades mannitol via 6-phosphogluconate as indicated in Fig. 1 has been presented (1, 9, 16, 17). As noted above, mannitol-grown cells of P. acidovorans have mannitol dehydrogenase and fructokinase. This readily available supply of fructose 6-phosphate may account for the low level of fructose-bisphosphatase in such cells. Moreover, these cells had elevated levels of glucosephosphate isomerase and glucose-6-phosphate dehydrogenase suggesting participation of the Entner-Doudoroff pathway (Fig. 1). However, phosphogluconate dehydratase and PKDG aldolase were not induced by growth on mannitol. Furthermore, a PKDG aldolase-deficient mutant that is unable to grow on gluconate nevertheless grows on mannitol (M. L. Rogers, unpublished data). A recently discovered alternate pathway for degradation of fructose (20) may provide a supplementary route for degradation of mannitol also. When grown on fructose, P. acidovorans and certain other aerobic pseudomonads possess phosphoenolpyruvate:fructose phosphotransferase and 1-phosphofructokinase, which constitute a modified EMP pathway in conjunction with fructose-bisphosphate aldolase. Evidence for the simultaneous participation of the Entner-Doudoroff and modified EMP pathways in degradation of fructose by P. acidovorans (20) and several other species (20, 26) has been obtained. Although phosphoenolpyruvate-dependent phosphorylation of fructose generally is believed to represent a transport system for fructose, this activity has been demonstrated only in crude extracts (17, 20). No evidence for an obligate coupling of phosphorylation to transport is available. Moreover, mannitol significantly induced phosphoenolpyruvate:fructose phosphotransferase and 1-phosphofructokinase in strains of P. aeruginosa that were able to oxidize mannitol to fructose (17). Phosphoenolpyruvate-dependent phosphorylation of intracellular fructose was excluded because mutants of P. aeruginosa, which were defective for glucosephosphate isomerase or glucose-6-phosphate dehydrogenase, were unable to grow on mannitol. However, this interpretation does not consider the possibility that inhibition of growth by accumulated hexosephosphate derivatives, which was demonstrated, could have obscured participation of a modified EMP pathway in degradation of mannitol. Therefore, the possible role of a modified EMP pathway in dissimilation of mannitol remains to be clarified. CARBOHYDRATE METABOLISM IN P. 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