316
Biochimica et Biophysica Acta, 538 (1978) 316--327 © Elsevier/North-Holland Biomedical Press
BBA 28405
MICROBIAL METABOLISM OF ALIPHATIC GLYCOLS BACTERIAL METABOLISM OF E T H Y L E N E GLYCOL
JANET CHILD and ANDREW WILLETTS
Department of Biological Sciences, University of Exeter, Perry Road, Exeter EX4 4QG (U.K.) (Received May 9th, 1977)
Summary A species of Flavobacterium isolated from pond water by its ability to grow aerobically on ethylene glycol as the role source of carbon initially oxidised the diol to glyoxylate via glycollate. The glyoxylate was metabolised by the glycerate pathway to acetyl-CoA. The acetyl-CoA was further metabolised by the tricarboxylic acid cycle plus malate synthase acting anaplerotically.
Introduction Ethylene glycol (ethane-l,2-diol) is widely distributed in the biosphere as a result of its use in various manufacturing processes, m o t o r car antifreeze preparations and deicing agents for aircraft and airfield runways. It is an established intermediate in the biodegradation of various polyethylene glycols [1], and may be formed during the biodegradation of some non-ionic detergents [2]. The biodegradation of ethylene glycol by activated sludge and river waters has been established at the phenomological level [3]. Species of several different genera of bacteria utilise ethylene glycol as the sole source of carbon and energy [4,5]. However, the microbial metabolism of ethylene glycol remains uncharacterized, although an ethylene glycol-metabolizing species of A z o t o bacter [6] was reported to metabolise glyoxylate to acetyl-CoA via the glycerate pathway [ 7]. The present paper describes the metabolism of ethylene glycol by a pure culture of a bacterium (NCIB 11171) selected for growth on ethylene glycol as the sole source of carbon.
Abbreviation: DCPIP, 2,6-dichlorophenolindophenol.
317 Materials and Methods
Microorganism, maintenance and culture. Microorganisms were isolated from soil and water samples in a minimal medium which was also employed for the batch culture of selected microorganisms. The medium contained (per 1 of distilled water) 2 g ethylene glycol plus the following mineral salts: 1 g KH2PO4; 1 g K2HPO4; 1 g KNO3; 1 g (NH4)2SO4; 0.1 g MgSO4 • 7H~O; 0.1 g NaC1; 20 pg CaCI~; 20 pg CuSO4 • 5H20; 20 pg MnSO4 • 5H20; 20 pg ZnSO4 • 7H20; 20 pg FeSO4" 7H20 and 20 pg (NH4)6Mo~O24" 4H20 adjusted to pH 7.0 and sterilised by autoclaving. The bacterium subsequently used in this investigation was selected from pond water; it was a yellow-pigmented, Gram-negative, non-motile rod identified as a species of Flavobacterium [8] and deposited with the National Culture of Industrial Bacteria, Torrey Research Station, Aberdeen, U.K. as NCIB 11171. NCIB 11171 was maintained on slopes of ethylene glycol growth medium solidified with 2.5% (w/v) Oxoid Agar No. 3. Growth on ethylene glycol was routinely carried out at 27°C. Liquid cultures were incubated for approximately 48 h on a rotary shaker, usually in 2-1 conical flasks containing 1 1 of growth medium inoculated with 100 ml of starter culture grown on minimal medium for 30 h. Preparation of washed suspensions. Bacterial cells were harvested (0°C, 10 000 × g, 20 min), resuspended in 0.1 M potassium phosphate buffer, pH 7.0 reharvested and either used immediately or stored at --15°C until required. Preparation of cell fractions. Bacteria in 0.1 M potassium phosphate buffer, pH 7.0, were disrupted ultrasonically at 0°C using an MSE ultrasonic disintegrator, operated at a frequency of 20 kHz with a maximum amplitude of 8--10 pm, for 5 min. The resultant preparation was centrifuged (0°C, 10 000 × g, 15 min), the supernatant solution used as a cell-free extract and the pellet re-suspended in 0.1 M potassium phosphate buffer, pH 7.0, and used as a particulate cellular preparation. Analytical methods. Protein was determined by a modified Biuret method [9]. A standard curve was prepared using bovine serum albumin. Oxalate was determined by titration in hot 2 M H2SO4 with 10 mM KMnO4 using 50.0-ml aliquots of reaction mixtures after removal of cells or cell fractions by centrifugation. 2.0 ml of a standard solution of sodium oxalate (10 mM) were added prior to titration and the amount of permanganate required to give a pink colour, in excess of that of an equivalent blank, was determined (1 ml 10 mM KMnO4 - 25 pmol oxalate). Ethylene glycol [10] and glyoxylate [11,12] were assayed eolorimetrically. Preparation of [U-14C]ethylene glycol. Uniformly labelled ethylene glycol (spec. act. 10.8 Ci/mol) was obtained from the Radiochemical Centre, Amersham, Bucks, U.K., and was subsequently purified by chromatography [13]. Metabolism of [U-14C]ethylene glycol. The incorporation of radioactivity from [U-~4C]ethylene glycol b y ethanol-soluble fractions of NCIB 11171 was assessed by established methods [14]. Manometric methods. Conventional manometric techniques [15] were used to measure respiration of washed cell suspension. Vessels received 5--30 mg dry
318 weight washed cells, 50 pmol potassium phosphate buffer, pH 7.0, and 30 ~mol substrate in a volume of 2.8 ml. The centre well contained 0.2 ml of 20% (w/v) NaOH. Oxygen consumption was measured at 25°C. CO2 production was measured at 25°C omitting NaOH from the centre well. Ethylene glycol-dependent oxygen uptake and glycollate-dependent oxygen uptake by both cell-free extracts and particulate cellular preparations were assayed manometrically. The main compartment contained 200 pmol Tris buffer, pH 8.0, plus 20 pmol ethylene glycol and sodium glycollate respectively. Enzyme assays. Spectrophotometric assays were performed on a Unicam SP 8000 spectrophotometer at 25°C. The amounts of enzymes assayed were such that rates were normally linear with respect to time for at least 3 min and were proportional to the amount of extract added. Malate dehydrogenase (decarboxylating) (EC 1.1.1.40) was assayed by measuring the NADP plus NAD-dependent decarboxylation of malate of pyruvate at 340 nm. The reaction mixture contained in 3.0 ml final volume: 75 pmol glycylglycine buffer, pH 7.4; 2 pmol sodium malate; 3 pmol MgC12; 0.3 ~mol NAD; 0.3 ~mol NADP and 0.1 ml cell-free extract containing approximately 0.2 mg protein. Malate dehydrogenase (EC 1.1.1.37) was assayed by measuring the NADHdependent reduction of oxaloacetate at 340 nm. The reaction mixture contained in 3.0 ml final volume: 75 pmol glycylglycine buffer, pH 7.4; 0.3 pmol sodium oxaloacetate; 0.15 pmol NADH and 0.001 ml cell-free extract containing approximately 2 pg protein. Hydroxypyruvate reductase (EC 1.1.1.29) was assayed by measuring the NADH-plus NADPH-dependent reduction of hydroxypyruvate at 340 nm. The reaction mixture contained in 3.0 ml final volume: 200 pmol potassium phosphate buffer, pH 7.0; 1 pmol lithium hydroxypyruvate; 0.3 pmol NADH, 0.3 pmol NADPH and 0.1 ml cell-free extract containing approx. 0.2 mg protein. The formation of oxaloacetate from phosphoenolpyruvate by phosphoenolpyruvate carboxylase (EC 4.1.1.31) was followed by reducing the oxaloacetate produced to malate by addition to the reaction mixture of an excess of malate dehydrogenase. The reaction mixture contained in 3.0ml final volume: pmol Tris buffer, pH 7.7; 0.3 pmol sodium phosphoenolpyruvate; 9 pmol MgC12; 10 pmol reduced glutathione; 0.3 gmol NADH; 150 pmol KHCO3; 5.5 units malate dehydrogenase and 0.1 ml cell-free extract. The carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase (EC 6.4.1.1) was also coupled to malate dehydrogenase. The reaction mixture contained in 3.0 ml final volume: 100 pmol Tris buffer, pH 7.2; 5 ~mol sodium pyruvate; 9 pmol MgC12; 0.3 pmol ATP; 0.3 gmol NADH; 20 pmol KHCO3; 5.5 units malate dehydrogenase and 0.1 ml cell-free extract. Catalase (EC 1.11.1.6) was assayed by measuring the decrease in hydrogen peroxide at 240 nm. Reaction mixtures contained in 3.0 ml final volume: 100 pmol potassium phosphate buffer, pH 7.0; 40 pmol H202 and 0.005 ml cellfree extract containing approx. 0.01 mg protein. Under these conditions, the extinction coefficient E240 for H202 was 4.36 • 104 mol-1/cm -1. Glycine aminotransferase (EC 2.6.1.4) was assayed chromatographically [ 17]. Reaction mixtures contained in 2.7 ml final volume: 200 pmol potassium phosphate buffer, pH 7.5; 20 pmol sodium glyoxylate; 30 pmol sodium gluta-
319 mate; 0.1 pmol pyridoxal phosphate and 0.1 ml cell-free extract containing approximately 0.2 mg protein. The reaction was started by addition of glyoxylate to otherwise complete reaction mixtures and was terminated after 15 min at 25°C by heating at 80°C for 5 min. To demonstrate the glycine formed during the reaction, the various reaction mixtures were evaporated to a small volume and subjected to chromatography on thin layer cellulose plates. The following solvent systems were used: (A) butanol/acetone/water (10 : 10 : 5, v/v); (B) phenol/water/formic acid (75 : 25 : 1, v/v). After drying, the plates were sprayed with 0.4% (w/v) ninhydrin in water-saturated butanol and heated at 105°C for 5 min. The following enzymes were assayed by published methods: glyoxylate reductase, EC 1.1.1.26 [18]; malate synthase, EC 4.1.3.2 [19]; isocitrate lyase, EC 4.1.3.1 [ 19] ; citrate (si)-synthase, EC 4.1.3.7 [20] ; pyruvate dehydrogenase [21] ; glyoxylate carboligase [11,22] ; glycerate kinase, EC 2.7.1.31 [23] ; pyruvate kinase, EC 2.7.1.40 [24]; erythro-/3-hydroxyaspartate dehydratase [25], erythro-/3-hydroxyaspartate aldolase [25]; glyoxylate dehydrogenase, EC 1.2.1.17 [26]; isocitrate dehydrogenase, EC 1.1.1.41 and EC 1.1.1.42 [27]; phosphoenolpyruvate carboxykinase (GTP) EC 4.1.1.32 [28]; succinate dehydrogenase, EC 1.3.99.1 [29]; tartronic semialdehyde reductase [33]; glycollate dehydrogenase [ 16]. Results
Growth studies Growth of NCIB 11171 in liquid culture on ethylene glycol as the sole source of carbon was optimum using an initial diol concentration of 30 mM. No growth occurred in mineral-salts medium alone. Best growth was obtained in well-aerated liquid culture; only poor growth was observed in static aerobic culture. The optimum initial pH of the minimal medium was pH 7.0. The optimum growth temperature was 30°C. There was no growth below 8°C and limited growth at 37°C. Inorganic nitrogen in the form of either NH~ or NO~, b u t not NO~, supported growth. There was no growth in the absence of exogenous fixed nitrogen. Under optimum conditions NCIB 11171 grew exponentially for approx. 24 h after a short lag phase. The mean doubling time of NCIB 11171 during exponential growth, as assessed turbimetrically, was approximately 3.4 h. NCIB 11171 was able to grow on a number of alcohols, carboxylic acids, esters and other diols (Table I). However, no growth occurred on di- or triethylene glycol. Growth was generally poor on carbohydrates and amino acids.
Identification of intermediates of ethylene glycol metabolism Samples of ethylene glycol minimal medium taken at various times after inoculation of NCIB 11171 were shown to contain up to a maximum of 5 nmol/ml of glyoxylate or a c o m p o u n d that have a phenylhydrazone with the same Xmax as glyoxylate. Glyoxylate accumulation by NCIB 11171 closely paralleled growth. Oxalate accumulated in resting cultures after exponential growth up to a maximum of 0.7 pmol/ml in 75 h.
320
Oxidation of substrates by washed suspensions Washed suspensions of ethylene glycol-grown cells when compared to equivalent succinate-grown cells were capable of rapid oxidation of ethylene glycol, glycolaldehyde and other primary alcohols such as ethanol, propanol and butanol (Table II). Glycollate and glyoxylate were also rapidly oxidised after a lag of approximately 4 min. A number of tricarboxylic acid cycle intermediates were readily oxidised. Oxalate and glycine were not oxidised. Washed suspensions of glycollate-grown cells were capable of rapid oxidation of glycollate and glyoxylate (Table II). Ethylene glycol and glycolaldehyde were oxidised less rapidly. The presence of 10 pM fluoroacetate inhibited the oxidation of a number of different substrates by washed suspensions of ethylene glycol-grown, glycollategrown and succinate-grown cells (Table III).
Incorporation of radioactivity from [U-14C]ethylene glycol by washed suspensions Washed suspensions of ethylene glycol-grown cells progressively incorporated radioactivity from [U-14C]ethylene glycol into the ethanol-soluble fraction. TABLE
I
GROWTH
OF NCIB 11171
ON SINGLE
CARBON
SOURCES
NCIB 11171 was grown on the mineral salts medium described in Materials and Methods supplemented w i t h a d d i t i o n s o f v a r i o u s s i n g l e c a r b o n s o u r c e s a t e i t h e r 3 0 m M o r 1 0 r a M , as i n d i c a t e d . G r o w t h w a s assessed as a f u n c t i o n o f t i m e u s i n g a P y e U n i c a m SP 1 8 0 0 s p e c t r o p h o t o m e t e r reading at 420 nm. Key to s y m b o l s : G r o w t h : + + + + + , e x c e l l e n t ; + + + + , v e r y g o o d ; + + + , g o o d ; + + , s o m e ; +, s l i g h t ; - - , n o n e w i t h i n 7 days.
Alcohols (at 30 mM) Ethanol Propan-l-ol
Mean gener-
Mean gener-
ation time (h)
ation time (h) Carboxyhydrates
++++ ++++
(at 30 mM)
3.20 3.27
D(--)-Fructose
+++++
2.66
D(--)-Glucose
--
--
Butan-l-ol
+++++
2.76
Maltose
--
--
Isobutanol Benzyl alcohol Ethylene glycol
++ -++++
6.83 -3.40
Lactose Sucrose
---
----
Propane-l,3-diol Butane-2,3-diol
+ +++++
13.78 2.72
Carboxylic
Butane-1,4-diol Glycerol
-++
-6.79
Acetate Propio nate
+++++ --
2.81 --
Isopropanol
+++
4.56
Butyrate Benzoate
+ --
Glycollate Glyoxylate
++++ +++
Oxalate Succinate Malate Pyruvate
+ +++++ +++++ +++++
Amino acids (at 30 raM) Glutamate Asparate Leucine Glycine, arginine, proline, valine, lysine, methionine, phenylalanine, serine, cysteine, histidine, alanine
++ + +
6.98 13.34 14.22
acids (at 30 mM)
12.98 -3.52 4.44 14.05 2.71 2.80 2.66
Esters and ether glycols (at 10 mM) --
Methyl formate Methyl acetate Ethylene glycol diacetate 2-Methoxyethanol Diethylene glycol Triethylene glycol
+ + +++ + ---
15.09 13.99 4.39 14.47 ---
321
T A B L E II OXIDATION OF SUBSTRATES BY WASHED SUSPENSIONS OF NCIB 11171 W a s h e d s u s p e n s i o n s o f cells o f N C I B 1 1 1 7 1 a f t e r g r o w t h o n t h e single c a r b o n s o u r c e s i n d i c a t e d w e r e prep a r e d a n d a s s a y e d f o r t h e a b i l i t y to o x i d i s e a v a r i e t y o f single s u b s t r a t e s b y m a n o m e t r y as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e rates g i v e n are c o r r e c t e d f o r e n d o g e n o u s o x i d a t i o n , n.t., n o t t e s t e d . V a l u e s are Q O 2 v a l u e s (pl 0 2 c o n s u m e d / h p e r m g d r y w t . cells). substrate for oxidation
E t h y l e n e glycolg r o w n cells
Glycollateg r o w n ceils
Succinateg r o w n cells
Ethylene glycol Glycolaldehyde Glycollate Glyoxylate Glyoxal Oxalate Acetate Succinate Fructose Glucose Glycine Ethanol Propan-l-ol Butan-l-ol Malate Citrate Isocitrate Oxaloacetate
30.7 30.6 19.6 16.0 17.0 0 25.5 25.7 13.9 0 1.0 27.3 23.8 27.3 26.8 30.6 29.9 26.4
10.0 16.8 34.0 33.0 8.4 0 35.6 36.4 n.t. n.t. n.t. n.t. n.t. n.t. 30.8 33.3 34.1 32.9
7.1 9.4 11.9 11.9 0 0 40.4 38.5 3.6 0 0.3 5.1 n.t. n.t. 36.2 40.1 37.8 41.6
The relative distribution of radioactivity among the various labelled metabolites in the ethanol-soluble fractions was analysed. In 1-min and 3-min samples the radioactivity was located almost entirely in glycollate.
Enzymes utilising ethylene glycol in cell fractions An ethylene glycol oxidase located exclusively in the particulate cellular preparation was induced by growth of NCIB 11171 on ethylene glycol but not glycollate (Table IV). The enzyme could not be solubilised by detergent treatment. Glyoxylate, but not oxalate, was detected in spent reaction mixtures. FMN, FAD, phenazine methosulphate and c y t o c h r o m e c were ineffective as electron acceptors. The enzyme was inhibited by a number of reagents (Table V). There was no evidence of either an ethylene glycol oxidase or dehydrogenase in cell-free extracts of NCIB 11171.
Enzymes utilising glycollate in cell fractions A glycollate oxidase located in the particulate cellular preparation was induced by growth of NCIB 11171 on glycollate (Table IV). The enzyme was also induced to a lesser extent by growth on ethylene glycol. Some glyoxylate but no oxalate was detected in spent reaction mixtures. FMN, FAD, phenazine methosulphate and cytochrome c were ineffective on the enzyme. The influence of a number of inhibitors (Table V) suggested that the enzyme was different from the particulate ethylene glycol oxidase of NCIB 11171. A DCPIP-dependent glycollate dehydrogenase located in the cell-free extract
322
o~
~o nD
Biochimica et Biophysica Acta, 538 (1978) 316--327 © Elsevier/North-Holland Biomedical Press
BBA 28405
MICROBIAL METABOLISM OF ALIPHATIC GLYCOLS BACTERIAL METABOLISM OF E T H Y L E N E GLYCOL
JANET CHILD and ANDREW WILLETTS
Department of Biological Sciences, University of Exeter, Perry Road, Exeter EX4 4QG (U.K.) (Received May 9th, 1977)
Summary A species of Flavobacterium isolated from pond water by its ability to grow aerobically on ethylene glycol as the role source of carbon initially oxidised the diol to glyoxylate via glycollate. The glyoxylate was metabolised by the glycerate pathway to acetyl-CoA. The acetyl-CoA was further metabolised by the tricarboxylic acid cycle plus malate synthase acting anaplerotically.
Introduction Ethylene glycol (ethane-l,2-diol) is widely distributed in the biosphere as a result of its use in various manufacturing processes, m o t o r car antifreeze preparations and deicing agents for aircraft and airfield runways. It is an established intermediate in the biodegradation of various polyethylene glycols [1], and may be formed during the biodegradation of some non-ionic detergents [2]. The biodegradation of ethylene glycol by activated sludge and river waters has been established at the phenomological level [3]. Species of several different genera of bacteria utilise ethylene glycol as the sole source of carbon and energy [4,5]. However, the microbial metabolism of ethylene glycol remains uncharacterized, although an ethylene glycol-metabolizing species of A z o t o bacter [6] was reported to metabolise glyoxylate to acetyl-CoA via the glycerate pathway [ 7]. The present paper describes the metabolism of ethylene glycol by a pure culture of a bacterium (NCIB 11171) selected for growth on ethylene glycol as the sole source of carbon.
Abbreviation: DCPIP, 2,6-dichlorophenolindophenol.
317 Materials and Methods
Microorganism, maintenance and culture. Microorganisms were isolated from soil and water samples in a minimal medium which was also employed for the batch culture of selected microorganisms. The medium contained (per 1 of distilled water) 2 g ethylene glycol plus the following mineral salts: 1 g KH2PO4; 1 g K2HPO4; 1 g KNO3; 1 g (NH4)2SO4; 0.1 g MgSO4 • 7H~O; 0.1 g NaC1; 20 pg CaCI~; 20 pg CuSO4 • 5H20; 20 pg MnSO4 • 5H20; 20 pg ZnSO4 • 7H20; 20 pg FeSO4" 7H20 and 20 pg (NH4)6Mo~O24" 4H20 adjusted to pH 7.0 and sterilised by autoclaving. The bacterium subsequently used in this investigation was selected from pond water; it was a yellow-pigmented, Gram-negative, non-motile rod identified as a species of Flavobacterium [8] and deposited with the National Culture of Industrial Bacteria, Torrey Research Station, Aberdeen, U.K. as NCIB 11171. NCIB 11171 was maintained on slopes of ethylene glycol growth medium solidified with 2.5% (w/v) Oxoid Agar No. 3. Growth on ethylene glycol was routinely carried out at 27°C. Liquid cultures were incubated for approximately 48 h on a rotary shaker, usually in 2-1 conical flasks containing 1 1 of growth medium inoculated with 100 ml of starter culture grown on minimal medium for 30 h. Preparation of washed suspensions. Bacterial cells were harvested (0°C, 10 000 × g, 20 min), resuspended in 0.1 M potassium phosphate buffer, pH 7.0 reharvested and either used immediately or stored at --15°C until required. Preparation of cell fractions. Bacteria in 0.1 M potassium phosphate buffer, pH 7.0, were disrupted ultrasonically at 0°C using an MSE ultrasonic disintegrator, operated at a frequency of 20 kHz with a maximum amplitude of 8--10 pm, for 5 min. The resultant preparation was centrifuged (0°C, 10 000 × g, 15 min), the supernatant solution used as a cell-free extract and the pellet re-suspended in 0.1 M potassium phosphate buffer, pH 7.0, and used as a particulate cellular preparation. Analytical methods. Protein was determined by a modified Biuret method [9]. A standard curve was prepared using bovine serum albumin. Oxalate was determined by titration in hot 2 M H2SO4 with 10 mM KMnO4 using 50.0-ml aliquots of reaction mixtures after removal of cells or cell fractions by centrifugation. 2.0 ml of a standard solution of sodium oxalate (10 mM) were added prior to titration and the amount of permanganate required to give a pink colour, in excess of that of an equivalent blank, was determined (1 ml 10 mM KMnO4 - 25 pmol oxalate). Ethylene glycol [10] and glyoxylate [11,12] were assayed eolorimetrically. Preparation of [U-14C]ethylene glycol. Uniformly labelled ethylene glycol (spec. act. 10.8 Ci/mol) was obtained from the Radiochemical Centre, Amersham, Bucks, U.K., and was subsequently purified by chromatography [13]. Metabolism of [U-14C]ethylene glycol. The incorporation of radioactivity from [U-~4C]ethylene glycol b y ethanol-soluble fractions of NCIB 11171 was assessed by established methods [14]. Manometric methods. Conventional manometric techniques [15] were used to measure respiration of washed cell suspension. Vessels received 5--30 mg dry
318 weight washed cells, 50 pmol potassium phosphate buffer, pH 7.0, and 30 ~mol substrate in a volume of 2.8 ml. The centre well contained 0.2 ml of 20% (w/v) NaOH. Oxygen consumption was measured at 25°C. CO2 production was measured at 25°C omitting NaOH from the centre well. Ethylene glycol-dependent oxygen uptake and glycollate-dependent oxygen uptake by both cell-free extracts and particulate cellular preparations were assayed manometrically. The main compartment contained 200 pmol Tris buffer, pH 8.0, plus 20 pmol ethylene glycol and sodium glycollate respectively. Enzyme assays. Spectrophotometric assays were performed on a Unicam SP 8000 spectrophotometer at 25°C. The amounts of enzymes assayed were such that rates were normally linear with respect to time for at least 3 min and were proportional to the amount of extract added. Malate dehydrogenase (decarboxylating) (EC 1.1.1.40) was assayed by measuring the NADP plus NAD-dependent decarboxylation of malate of pyruvate at 340 nm. The reaction mixture contained in 3.0 ml final volume: 75 pmol glycylglycine buffer, pH 7.4; 2 pmol sodium malate; 3 pmol MgC12; 0.3 ~mol NAD; 0.3 ~mol NADP and 0.1 ml cell-free extract containing approximately 0.2 mg protein. Malate dehydrogenase (EC 1.1.1.37) was assayed by measuring the NADHdependent reduction of oxaloacetate at 340 nm. The reaction mixture contained in 3.0 ml final volume: 75 pmol glycylglycine buffer, pH 7.4; 0.3 pmol sodium oxaloacetate; 0.15 pmol NADH and 0.001 ml cell-free extract containing approximately 2 pg protein. Hydroxypyruvate reductase (EC 1.1.1.29) was assayed by measuring the NADH-plus NADPH-dependent reduction of hydroxypyruvate at 340 nm. The reaction mixture contained in 3.0 ml final volume: 200 pmol potassium phosphate buffer, pH 7.0; 1 pmol lithium hydroxypyruvate; 0.3 pmol NADH, 0.3 pmol NADPH and 0.1 ml cell-free extract containing approx. 0.2 mg protein. The formation of oxaloacetate from phosphoenolpyruvate by phosphoenolpyruvate carboxylase (EC 4.1.1.31) was followed by reducing the oxaloacetate produced to malate by addition to the reaction mixture of an excess of malate dehydrogenase. The reaction mixture contained in 3.0ml final volume: pmol Tris buffer, pH 7.7; 0.3 pmol sodium phosphoenolpyruvate; 9 pmol MgC12; 10 pmol reduced glutathione; 0.3 gmol NADH; 150 pmol KHCO3; 5.5 units malate dehydrogenase and 0.1 ml cell-free extract. The carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase (EC 6.4.1.1) was also coupled to malate dehydrogenase. The reaction mixture contained in 3.0 ml final volume: 100 pmol Tris buffer, pH 7.2; 5 ~mol sodium pyruvate; 9 pmol MgC12; 0.3 pmol ATP; 0.3 gmol NADH; 20 pmol KHCO3; 5.5 units malate dehydrogenase and 0.1 ml cell-free extract. Catalase (EC 1.11.1.6) was assayed by measuring the decrease in hydrogen peroxide at 240 nm. Reaction mixtures contained in 3.0 ml final volume: 100 pmol potassium phosphate buffer, pH 7.0; 40 pmol H202 and 0.005 ml cellfree extract containing approx. 0.01 mg protein. Under these conditions, the extinction coefficient E240 for H202 was 4.36 • 104 mol-1/cm -1. Glycine aminotransferase (EC 2.6.1.4) was assayed chromatographically [ 17]. Reaction mixtures contained in 2.7 ml final volume: 200 pmol potassium phosphate buffer, pH 7.5; 20 pmol sodium glyoxylate; 30 pmol sodium gluta-
319 mate; 0.1 pmol pyridoxal phosphate and 0.1 ml cell-free extract containing approximately 0.2 mg protein. The reaction was started by addition of glyoxylate to otherwise complete reaction mixtures and was terminated after 15 min at 25°C by heating at 80°C for 5 min. To demonstrate the glycine formed during the reaction, the various reaction mixtures were evaporated to a small volume and subjected to chromatography on thin layer cellulose plates. The following solvent systems were used: (A) butanol/acetone/water (10 : 10 : 5, v/v); (B) phenol/water/formic acid (75 : 25 : 1, v/v). After drying, the plates were sprayed with 0.4% (w/v) ninhydrin in water-saturated butanol and heated at 105°C for 5 min. The following enzymes were assayed by published methods: glyoxylate reductase, EC 1.1.1.26 [18]; malate synthase, EC 4.1.3.2 [19]; isocitrate lyase, EC 4.1.3.1 [ 19] ; citrate (si)-synthase, EC 4.1.3.7 [20] ; pyruvate dehydrogenase [21] ; glyoxylate carboligase [11,22] ; glycerate kinase, EC 2.7.1.31 [23] ; pyruvate kinase, EC 2.7.1.40 [24]; erythro-/3-hydroxyaspartate dehydratase [25], erythro-/3-hydroxyaspartate aldolase [25]; glyoxylate dehydrogenase, EC 1.2.1.17 [26]; isocitrate dehydrogenase, EC 1.1.1.41 and EC 1.1.1.42 [27]; phosphoenolpyruvate carboxykinase (GTP) EC 4.1.1.32 [28]; succinate dehydrogenase, EC 1.3.99.1 [29]; tartronic semialdehyde reductase [33]; glycollate dehydrogenase [ 16]. Results
Growth studies Growth of NCIB 11171 in liquid culture on ethylene glycol as the sole source of carbon was optimum using an initial diol concentration of 30 mM. No growth occurred in mineral-salts medium alone. Best growth was obtained in well-aerated liquid culture; only poor growth was observed in static aerobic culture. The optimum initial pH of the minimal medium was pH 7.0. The optimum growth temperature was 30°C. There was no growth below 8°C and limited growth at 37°C. Inorganic nitrogen in the form of either NH~ or NO~, b u t not NO~, supported growth. There was no growth in the absence of exogenous fixed nitrogen. Under optimum conditions NCIB 11171 grew exponentially for approx. 24 h after a short lag phase. The mean doubling time of NCIB 11171 during exponential growth, as assessed turbimetrically, was approximately 3.4 h. NCIB 11171 was able to grow on a number of alcohols, carboxylic acids, esters and other diols (Table I). However, no growth occurred on di- or triethylene glycol. Growth was generally poor on carbohydrates and amino acids.
Identification of intermediates of ethylene glycol metabolism Samples of ethylene glycol minimal medium taken at various times after inoculation of NCIB 11171 were shown to contain up to a maximum of 5 nmol/ml of glyoxylate or a c o m p o u n d that have a phenylhydrazone with the same Xmax as glyoxylate. Glyoxylate accumulation by NCIB 11171 closely paralleled growth. Oxalate accumulated in resting cultures after exponential growth up to a maximum of 0.7 pmol/ml in 75 h.
320
Oxidation of substrates by washed suspensions Washed suspensions of ethylene glycol-grown cells when compared to equivalent succinate-grown cells were capable of rapid oxidation of ethylene glycol, glycolaldehyde and other primary alcohols such as ethanol, propanol and butanol (Table II). Glycollate and glyoxylate were also rapidly oxidised after a lag of approximately 4 min. A number of tricarboxylic acid cycle intermediates were readily oxidised. Oxalate and glycine were not oxidised. Washed suspensions of glycollate-grown cells were capable of rapid oxidation of glycollate and glyoxylate (Table II). Ethylene glycol and glycolaldehyde were oxidised less rapidly. The presence of 10 pM fluoroacetate inhibited the oxidation of a number of different substrates by washed suspensions of ethylene glycol-grown, glycollategrown and succinate-grown cells (Table III).
Incorporation of radioactivity from [U-14C]ethylene glycol by washed suspensions Washed suspensions of ethylene glycol-grown cells progressively incorporated radioactivity from [U-14C]ethylene glycol into the ethanol-soluble fraction. TABLE
I
GROWTH
OF NCIB 11171
ON SINGLE
CARBON
SOURCES
NCIB 11171 was grown on the mineral salts medium described in Materials and Methods supplemented w i t h a d d i t i o n s o f v a r i o u s s i n g l e c a r b o n s o u r c e s a t e i t h e r 3 0 m M o r 1 0 r a M , as i n d i c a t e d . G r o w t h w a s assessed as a f u n c t i o n o f t i m e u s i n g a P y e U n i c a m SP 1 8 0 0 s p e c t r o p h o t o m e t e r reading at 420 nm. Key to s y m b o l s : G r o w t h : + + + + + , e x c e l l e n t ; + + + + , v e r y g o o d ; + + + , g o o d ; + + , s o m e ; +, s l i g h t ; - - , n o n e w i t h i n 7 days.
Alcohols (at 30 mM) Ethanol Propan-l-ol
Mean gener-
Mean gener-
ation time (h)
ation time (h) Carboxyhydrates
++++ ++++
(at 30 mM)
3.20 3.27
D(--)-Fructose
+++++
2.66
D(--)-Glucose
--
--
Butan-l-ol
+++++
2.76
Maltose
--
--
Isobutanol Benzyl alcohol Ethylene glycol
++ -++++
6.83 -3.40
Lactose Sucrose
---
----
Propane-l,3-diol Butane-2,3-diol
+ +++++
13.78 2.72
Carboxylic
Butane-1,4-diol Glycerol
-++
-6.79
Acetate Propio nate
+++++ --
2.81 --
Isopropanol
+++
4.56
Butyrate Benzoate
+ --
Glycollate Glyoxylate
++++ +++
Oxalate Succinate Malate Pyruvate
+ +++++ +++++ +++++
Amino acids (at 30 raM) Glutamate Asparate Leucine Glycine, arginine, proline, valine, lysine, methionine, phenylalanine, serine, cysteine, histidine, alanine
++ + +
6.98 13.34 14.22
acids (at 30 mM)
12.98 -3.52 4.44 14.05 2.71 2.80 2.66
Esters and ether glycols (at 10 mM) --
Methyl formate Methyl acetate Ethylene glycol diacetate 2-Methoxyethanol Diethylene glycol Triethylene glycol
+ + +++ + ---
15.09 13.99 4.39 14.47 ---
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T A B L E II OXIDATION OF SUBSTRATES BY WASHED SUSPENSIONS OF NCIB 11171 W a s h e d s u s p e n s i o n s o f cells o f N C I B 1 1 1 7 1 a f t e r g r o w t h o n t h e single c a r b o n s o u r c e s i n d i c a t e d w e r e prep a r e d a n d a s s a y e d f o r t h e a b i l i t y to o x i d i s e a v a r i e t y o f single s u b s t r a t e s b y m a n o m e t r y as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e rates g i v e n are c o r r e c t e d f o r e n d o g e n o u s o x i d a t i o n , n.t., n o t t e s t e d . V a l u e s are Q O 2 v a l u e s (pl 0 2 c o n s u m e d / h p e r m g d r y w t . cells). substrate for oxidation
E t h y l e n e glycolg r o w n cells
Glycollateg r o w n ceils
Succinateg r o w n cells
Ethylene glycol Glycolaldehyde Glycollate Glyoxylate Glyoxal Oxalate Acetate Succinate Fructose Glucose Glycine Ethanol Propan-l-ol Butan-l-ol Malate Citrate Isocitrate Oxaloacetate
30.7 30.6 19.6 16.0 17.0 0 25.5 25.7 13.9 0 1.0 27.3 23.8 27.3 26.8 30.6 29.9 26.4
10.0 16.8 34.0 33.0 8.4 0 35.6 36.4 n.t. n.t. n.t. n.t. n.t. n.t. 30.8 33.3 34.1 32.9
7.1 9.4 11.9 11.9 0 0 40.4 38.5 3.6 0 0.3 5.1 n.t. n.t. 36.2 40.1 37.8 41.6
The relative distribution of radioactivity among the various labelled metabolites in the ethanol-soluble fractions was analysed. In 1-min and 3-min samples the radioactivity was located almost entirely in glycollate.
Enzymes utilising ethylene glycol in cell fractions An ethylene glycol oxidase located exclusively in the particulate cellular preparation was induced by growth of NCIB 11171 on ethylene glycol but not glycollate (Table IV). The enzyme could not be solubilised by detergent treatment. Glyoxylate, but not oxalate, was detected in spent reaction mixtures. FMN, FAD, phenazine methosulphate and c y t o c h r o m e c were ineffective as electron acceptors. The enzyme was inhibited by a number of reagents (Table V). There was no evidence of either an ethylene glycol oxidase or dehydrogenase in cell-free extracts of NCIB 11171.
Enzymes utilising glycollate in cell fractions A glycollate oxidase located in the particulate cellular preparation was induced by growth of NCIB 11171 on glycollate (Table IV). The enzyme was also induced to a lesser extent by growth on ethylene glycol. Some glyoxylate but no oxalate was detected in spent reaction mixtures. FMN, FAD, phenazine methosulphate and cytochrome c were ineffective on the enzyme. The influence of a number of inhibitors (Table V) suggested that the enzyme was different from the particulate ethylene glycol oxidase of NCIB 11171. A DCPIP-dependent glycollate dehydrogenase located in the cell-free extract
322
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