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Inducible uptake and metabolism of glucose by the phosphorylative pathway in Pseudomonas putida CSV86

Aditya Basu, Prashant S. Phale
DOI: http://dx.doi.org/10.1111/j.1574-6968.2006.00285.x 311-316 First published online: 1 June 2006

Abstract

Pseudomonas putida CSV86 utilizes glucose, naphthalene, methylnaphthalene, benzyl alcohol and benzoate as the sole source of carbon and energy. Compared with glucose, cells grew faster on aromatic compounds as well as on organic acids. The organism failed to grow on gluconate, 2-ketogluconate, fructose and mannitol. Whole-cell oxygen uptake, enzyme activity and metabolic studies suggest that in strain CSV86 glucose utilization is exclusively by the intracellular phosphorylative pathway, while in Stenotrophomonas maltophilia CSV89 and P. putida KT2442 glucose is metabolized by both direct oxidative and indirect phosphorylative pathways. Cells grown on glucose showed five- to sixfold higher activity of glucose-6-phosphate dehydrogenase compared with cells grown on aromatic compounds or organic acids as the carbon source. Study of [14C]glucose uptake by whole cells indicates that the glucose is taken up by active transport. Metabolic and transport studies clearly demonstrate that glucose metabolism is suppressed when strain CSV86 is grown on aromatic compounds or organic acids.

Keywords
  • Pseudomonas
  • glucose metabolism
  • transport
  • enzyme induction
  • aromatic compound catabolism

Introduction

A variety of aromatic compounds are present in fresh and marine waters and in agricultural lands. The most effective and economical way to remove these compounds from the environment is by microbial degradation (Alexander, 1981). The bottleneck in the efficient degradation of these compounds is the presence of simple carbon sources like organic acids and sugars, which are preferentially utilized by microorganisms, and unless the simpler carbon sources are completely depleted, the toxic aromatic compounds are not degraded (Collier , 1996). In pseudomonads, glucose utilization follows two routes: (i) the direct oxidative pathway, which converts glucose to gluconate, 2-ketogluconate and then subsequently to 6-phosphogluconate by extracellular, high affinity, glucose dehydrogenase and gluconate dehydrogenase (Quay , 1972; Midgley & Dawes, 1973; Roberts , 1973; Lessie , 1979), and (ii) the intracellular, low affinity, nucleotide-dependent phosphorylative pathway (Lessie & Neidhardt, 1967; Tiwari & Campbell, 1969; Eisenberg , 1974; Guymon & Eagon, 1974) wherein glucose is converted to 6-phosphogluoconate by glucokinase and glucose 6-phosphate dehydrogenase (Zwf). The key intermediate for both pathways is 6-phosphogluconate, which enters the TCA cycle via glyceraldehyde-3-phosphate and pyruvate through the Entner-Doudoroff pathway (Fig. 1a) (Entner & Doudoroff, 1952; Tiwari & Campbell, 1969). Depending on the physiological conditions, one or other of the pathways predominates (Lessie & Phibbs, 1984).

Figure 1

Metabolic pathways involved in the utilization of glucose by (a) pseudomonads and (b) Pseudomonas putida CSV86. The enzymes involved are: Gcd, Gad: glucose and gluconate oxidase; Gct, Kgt: glucose- and 2-ketogluconate-transporter; Gck, Gnk, Kgk: glucose-, gluconate- and 2-ketogluconate-kinase; Zwf: glucose 6-phosphate dehydrogenase; Kgr: 2-keto 6-phospho gluconate reductase; Pgd: 6-phosphogluconate dehydratase; Kdga: 2-Keto-3-deoxy-6-phospho gluconate aldolase Cross indicates the inability of strain to utilize gluconate and 2-ketogluconate as carbon source.

It has been reported that degradation of aromatic compounds is repressed by glucose as well as by organic acids (Worsey & Williams, 1975; Duetz , 1994; Holtel , 1994; Schleissner , 1994; Muller , 1996, 1997; McFall , 1997). Such preferential utilization of simple carbon sources represses degradation of recalcitrant compounds in nature (referred to as carbon catabolite repression). Attempts have been made to overcome this repression by generating mutants defective in glucose utilization which will mineralize complex carbon sources like naphthalene efficiently even in the presence of glucose (Samanta , 2001).

Pseudomonas putida CSV86 has been shown to metabolize aromatic compounds preferentially over glucose and cometabolize aromatic compounds and organic acids (Basu , 2006). Here we report that strain CSV86 utilizes glucose only by the intracellular, phosphorylative pathway. The strain utilizes aromatic compounds and organic acids faster compared with glucose, and the glucose-metabolizing enzyme, Zwf, and glucose transport are inducible and suppressed by growth on aromatics or organic acids.

Materials and methods

Growth conditions

Bacterial cultures used in this study were P. putida CSV86 (Mahajan , 1994), Stenotrophomonas maltophilia CSV89 (Phale , 1995) and P. putida KT2442. Cultures were grown in 150 mL mineral salt medium [MSM; Basu , 2003] at 30°C on a rotary shaker (200 r.p.m.). The medium was supplemented aseptically with appropriate amounts of aromatic compounds (0.1%), glucose (0.25%) or organic acids (0.25%) as carbon source. Growth was monitored spectrophotometrically at 540 nm.

Metabolism of glucose

Glucose grown mid-log phase cells (∼200 mg) were harvested, washed twice with sterile distilled water, suspended in 50 mL of 10 mM glucose prepared in sterile distilled water and incubated at 30°C for 3.5 h. The pH of the suspension was constantly monitored and maintained at pH 7.0 with KOH (6 M). After incubation, the cells were removed by centrifugation. The supernatant was filtered through a 0.2 μm filter and lyophilized to a dry powder. Products formed from glucose were resolved by TLC (one dimension) and detected as described (Pujol & Kado, 2000).

Whole-cell oxygen uptake

Mid-log phase cells grown on the appropriate carbon source were used. Respiration rates were measured at 30°C using oxygraph (Hansatech, UK) fitted with a Clark-type O2 electrode as described earlier (Basu , 2003). Respiration rates were corrected for endogenous O2 consumption and expressed as nmol O2 consumed min−1 mg−1 of cells (wet weight).

Preparation of cell-free extracts and enzyme assays

Cell-free extracts were prepared as described earlier (Basu , 2003). Protein was estimated using folin-phenol reagent (Lowry , 1951). Glucose dehydrogenase (Matsushita & Ameyama, 1982), gluconate dehydrogenase (Matsushita , 1982) and glucose-6-phosphate dehydrogenase (Lessmann , 1975) activities were monitored as described. Enzyme activities are expressed either as nanomoles of substrate consumed or product formed or NADH formed or consumed per min. Specific activities are expressed as nmoles per min per mg of protein.

[14C]Glucose uptake, binding assay

Uptake of [U-14C]glucose was studied by modifying the method described previously (Sly , 1993). Cells were grown till late-log phase, harvested, washed twice and resuspended in MSM to an absorbance of 0.20 at 540 nm. Cell suspension was incubated at 30°C for 10 min in a shaking water bath. To the prewarmed cell suspension (10 mL), 5 nmol of [14C]glucose (BRIT, India, sp. ac. 140 mCi mmol−1) was added, and samples (100 μL) were withdrawn and rapidly filtered through 0.45 μm cellulose ester filters (Pall). The filters were immediately washed twice with sterile MSM (1 mL), air dried and vigorously mixed in scintillation cocktail (0.4% PPO and 0.025% POPOP in toluene). Radioactivity was measured using a liquid scintillation counter (Rackbeta LKB1209) and expressed as pmoles [14C]glucose accumulated.

All the experiments were performed at least three times in triplicate and the observed standard deviation was less than 5%.

Results and discussion

Pseudomonas putida CSV86 metabolizes naphthalene via the catechol meta-cleavage pathway, and benzyl alcohol via the catechol ortho-cleavage pathway (Mahajan , 1994; Basu , 2003). It also utilizes benzoic acid, and p-and o-hydroxy benzoic acid and p-and o-hydroxybenzyl alcohols (Basu , 2003). Besides aromatic compounds, strain CSV86 also utilizes glucose and glycerol, but it failed to grow on gluconate, 2-ketogluconate, fructose and mannitol. The growth profile on various carbon sources is shown in Fig 2. The observed specific growth rate (μ, h−1) and doubling time (h) on various carbon sources were found to be: naphthalene (0.51, 1.36), salicylate (0.34, 2.04), benzyl alcohol (0.54, 1.28), benzoate (0.59, 1.17) glucose (0.22, 3.15) and succinate (0.76, 0.91). Growth profile and kinetic analysis clearly demonstrated that CSV86 utilized aromatic compounds and organic acids faster than glucose. The observed μ and doubling time values on glucose for strains S. maltophilia CSV89 and P. putida KT2442 were 0.52 and 1.33; and 0.55 and 1.26, respectively, indicating that strain CSV86 utilizes glucose at a lower rate.

Figure 2

Growth profile of Pseudomonas putida CSV86 on naphthalene (○), salicylate (Graphic), benzyl alcohol (□), benzoate (⋄), glucose (▵) and succinate (Graphic).

To elucidate the glucose metabolic pathway, O2 uptake and enzyme activity studies were carried out. Strain CSV86 grown on glucose showed O2 uptake with glucose but failed to respire on gluconate and 2-ketogluconate. Naphthalene- and succinate-grown cells showed poor O2 uptake when incubated with glucose. Strains CSV89 and KT2442 showed O2 uptake in the presence of glucose, gluconate and 2-ketogluconate (Table 1). These results suggest that, unlike the other strains, CSV86 does not have the ability to metabolize gluconate and 2-ketogluconate. These observations were supported by measurements of enzyme activities and analysis of the products formed during metabolism of glucose. Specific activities for various enzymes involved in glucose metabolism are depicted in Table 2. Cell-free extracts prepared from all three strains showed activity of Zwf. Compared with glucose-, succinate- and naphthalene-grown cells of CSV86 showed five- to six-fold lower activity of Zwf, suggesting that the enzyme is inducible. Strain CSV86 showed activity of glucose oxidase but failed to show activity of gluconate oxidase, while strains CSV89 and KT2442 showed activity for both enzymes. TLC analysis of metabolites produced during metabolism of glucose by CSV86 detected very little gluconate and no 2-ketogluconate; however, strains CSV89 and KT2442 showed spots corresponding to gluconate (Rf 0.18, light blue) and 2-ketogluconate (Rf 0.18, dark brown), data not shown. Therefore, TLC analysis, whole-cell respiration and enzyme activity studies suggest that CSV86 does not metabolize glucose via gluconate and 2-ketogluconate, thus indicating the absence of the direct oxidative pathway. However, the presence of Zwf activity suggests that CSV86 utilizes glucose by the phosphorylative pathway. Based on these results, the proposed pathway for glucose metabolism in P. putida CSV86 is shown in Fig. 1b. In strains CSV89 and KT2442, both the phosphorylative and direct oxidative pathways appear to be operational for glucose metabolism (Fig. 1a).

View this table:
Table 1

Oxygen uptake rates with various substrates for Pseudomonas putida CSV86, Stenotrophomonas maltophilia CSV89 and P. putida KT2442

O2 uptake rates (nmol min−1 mg−1)
Pp CSV86Sm CSV89Pp KT2442
SubstrateGlcNaphSucGlcGlc
Glucose2.310.40.34.934.12
GluconateNDNDND0.784.61
2-KetogluconateNDNDND0.371.0
  • * Oxygen uptake has been corrected for endogenous cell respiration.

  • P. putida CSV86.

  • S. maltophilia CSV89.

  • § P. putida KT2442.

  • Cells were grown on: Naph, Naphthalene; Glc, Glucose; Suc, Succinate.

  • ND, not detected by this method.

View this table:
Table 2

Specific activities of glucose metabolizing enzymes in Pseudomonas putida CSV86 grown on naphthalene (0.1%) and glucose (0.25%), and Stenotrophomonas maltophilia CSV89 and P. putida KT2442 grown on glucose (0.25%)

Specific activity (nmol min−1 mg−1 protein)
Pp CSV86
EnzymeNaphGlcSuc Sm CSV89 Pp KT2442
Glucose oxidase1315.71417.116
Gluconate oxidaseNDNDND285.925
Glucose-6-phosphate dehydrogenase (Zwf)1463.41195.1170
  • ND, not detected.

To study glucose transport, [14C]glucose uptake by cells grown under different conditions was monitored. Glucose-grown CSV86 cells showed high rates of glucose uptake (Fig. 3). Addition of sodium azide (25 mM) after 1 min, inhibited glucose uptake, and cells preincubated for 10 min with sodium azide (25 mM) or formaldehyde (25 mM) did not show any uptake. These results demonstrate that glucose uptake in CSV86 is by active transport. Cells grown on succinic acid or naphthalene alone showed very low glucose uptake compared with glucose-grown cells. Similar results were observed when cells were grown on benzyl alcohol or pyruvate (data not shown). These results suggest that glucose transport in CSV86 is suppressed when cells are grown on organic acids or aromatic compounds.

Figure 3

[14C]Glucose uptake by Pseudomonas putida CSV86 cells grown on naphthalene (○), succinate (▿) and glucose (□). Inhibition of [14C]glucose uptake was observed when cells were exposed to sodium azide (25 mM) after one min (arrow,◊) or pretreated for 10 min with sodium azide (25 mM,) or formaldehyde (25 mM, ★).

Metabolic studies clearly demonstrated that glucose is metabolized in P. putida CSV86 by the phosphorylative pathway and not by the direct oxidative pathway. It has been shown that pseudomonads sequester glucose as gluconate and under glucose limitation conditions gluconate is utilized as the carbon source (Schleissner , 1997). However, this is not the case with strain CSV86 as it failed to accumulate gluconate in the medium, and did not utilize it as a carbon source. Although a very small amount of gluconate was formed during glucose metabolism, the inability to utilize it as the carbon source could be due to either lack of gluconate transport or gluconate metabolizing enzymes, or both. CSV86 utilizes glucose at slower rate compared with aromatic compounds and organic acids. The slow utilization of glucose may be due to regulation of glucose metabolizing enzymes and/or the transport process. In strain CSV86 the activity of Zwf was found to be inducible; five- to six-fold higher activity was observed when cells were grown on glucose compared with naphthalene and succinate. The Zwf activity was 35% and 65% lower in CSV86 compared with strains CSV89 and KT2442, respectively. As reported for other pseudomonads (Midgley & Dawes, 1973), glucose transport in CSV86 was sensitive to sodium azide and formaldehyde demonstrating the active transport of glucose, while significantly reduced glucose uptake by cells grown on aromatic compounds and organic acid indicates that this transport system is also inducible. Based on these results, we conclude that in P. putida CSV86 the metabolism of glucose is regulated at least at enzyme and transport level. The absence of the direct oxidative pathway and the low activity of Zwf in CSV86 may be responsible for the slow growth rate on glucose. Few strains have been previously reported to have only the phosphorylative pathway or to be defective in glucose metabolism (Lessie & Phibbs, 1984).

It has been reported that glucose and organic acid repress the utilization of various aromatic compounds by pseudomonads (Holtel , 1994; Schleissner , 1994; Muller , 1996; Rentz , 2004) and attempts have been made to improve the utilization of aromatic compounds in the presence of glucose by conjugal transfer of naphthalene and salicylate degradation genes into P. putida strains generated by mutagenesis which are deficient in glucose metabolism (Samanta , 2001). Such strains would utilize aromatic compounds with a higher specific growth rate compared with glucose and hence would be an asset for bioremediation. However, the viability of such strains in the environment is not known. Pseudomonas putida CSV86 is a natural isolate and grows on aromatic compounds with a higher specific growth rate than on glucose. It also showed a preference for aromatic compounds over glucose and cometabolizes aromatic compounds and organic acids (Basu , 2006). These unique properties, coupled with further characterization and subtle modification, may make this strain a potential candidate for bioremediation and environmental clean up.

Acknowledgements

A. B. thanks the University Grants Commission, India for the award of a senior research fellowship.

References

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