/ . Biochem., 78, 307-315 (1975)

Effect of Nitrate Reduction on the Enzyme Levels in Carbon Metabolism in Escherichia coli1 Isamu YAMAMOTO and Makoto ISHIMOTO Department of Chemical Microbiology, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060 Received for publication, January 7, 1975

The activities of twelve enzymes were measured in crude extracts from cells of Escherichia coli K-10 grown aerobically or anaerobically in a defined medium in the presence or absence of nitrate. The activities of isocitrate dehydrogenase, aconitate hydratase, 2-oxoglutarate dehydrogenase, malate dehydrogenase, malic enzyme, and D-lactate dehydrogenase (NAD+-independent) were found to be higher in cells grown in nitrate respiration than in those in fermentation, but lower than in those in respiration. This finding may explain the incomplete oxidation in nitrate respiration and, on the other hand, suggests the operation of the tricarboxylic acid cycle even under these conditions. The activities of succinate dehydrogenase and alcohol dehydrogenase in relation to the formation of fermentation product were as high in cells grown in fermentation as in those in respiration and were low in those in nitrate respiration. However, the ratio of the activities in the latter case to the activities in respiration was the same as the ratio for most enzymes in the tricarboxylic acid cycle. The level of lactate dehydrogenase (NAD+-dependent) was not affected by nitrate respiration but its activity in the extract was inhibited by nitrate and nitrite. The absence of lactate in the anaerobic culture with nitrate may be due to this inhibition as well as NADH oxidation by nitrate. Levels of glucose-6phosphate dehydrogenase and glutamate dehydrogenase were not altered by the growth conditions and that of pyruvate dehydrogenase was low only in cells grovyn in fermentation.

Many bacteria anaerobically oxidize various substances with nitrate in place of molecular oxygen and obtain energy from substrates which are not utilizable in fermentation. This . _,. ™ . . . . . , 1 This study was supported in part by a grant for Scientific Research from the Ministry of Education of Japan. Abbreviations: APAD+, 3-acetylpyridine adenine dinucleotide; TPP, thiamine pyrophosphate. Vol. 78, No. 2, 1975

process is called nitrate respiration, because cytochrome participates in the reduction (7). Complete oxidation of glucose to carbon dioxide was observed in denitrifying bacteria (2), but not in Aerobacter aerogenes (3), or Escherichia coli (4). The formation of hydrov J ' S e n a s e a n d formate hydrogenlyase is known to be inhibited in the presence of nitrate as well as oxygen (5). There are only a few reports on enzyme activities involved in the

307

I. YAMAMOTO and M. ISHIMOTO

308

carbon metabolism of cells under the conditions of nitrate respiration in comparison with those of respiration and fermentation. Wimpenny and Cole (6) reported that aconitate hydratase and fumarate hydratase were deficient in cells of E. coli grown anaerobically in the presence of nitrate and suggested an alteration of the pathways of pyruvate degradation from dehydrogenation to formate release, as well as the abolition of the tricarboxylic acid cycle in nitrate respiration. Takahara and Ishimoto (7) found low succinate dehydrogenase activity in cells of the bacterium grown under the conditions of nitrate respiration. In the present communication, the activities of twelve enzymes in carbon metabolism were compared in extracts from cells of E. coli K-10 grown aerobically or anaerobically in the presence or absence of nitrate. The activities of most of the enzymes, including those in the tricarboxylic acid cycle, in cells grown in nitrate respiration were between those found in cells grown aerobically and anaerobically in the absence of nitrate. The activities of succinate dehydrogenase and alcohol dehydrogenase were exceptionally low in the cells grown anaerobically in the presence of nitrate. An inhibitory effect of nitrate and nitrite on lactate dehydrogenase (NAD+-dependent) is also reported. EXPERIMENTAL PROCEDURES Cultivation of Bacteria — Escherichia coli K-10 Hfr was used in all experiments. A complex medium containing 10 g of meat extract (Wako), 10 g of polypeptone (Daigo Eiyo), and 2 g of NaCl per liter was inoculated with 2.5% volume of 9-hr culture in the same medium. After incubation for 9 hr at 37°, 50 ml of the resulting culture was transferred to 2 liters of a defined medium (6) containing: 4 g of glucose, 2.7 g of NH«C1, 0.55 g of KH..PO,, 6.3 g of K2HPO4, and 5 ml of mineral solution per liter (pH 7.2—7.4). Glucose was sterilized separately. The mineral solution contained 1 g of MgSO4-7H2O, 0.1 g of MnCl2-4H2O, 40 mg of FeSO4-7H2O, and 10 mg of CaCl2 per 100 ml and was acidified with H2SO4 to pH 2.

KNO3, 4 g per liter, was added to the medium, where indicated. Aerobic growth was maintained by rotary shaking of a 2-liter culture in a 5-liter Erlenmeyer flask at 200 rpm and 30° for 6 hr. For anaerobic growth, culture flasks were placed in an anaerobic pot and incubated at 37° for 8 hr under nitrogen. Cell growth was observed by measuring the absorbance of the culture at 660 nm in a 10 mm cuvette using a spectrophotometer (Hitachi 139). One unit of absorbance corresponds to a cell concentration of 0.40 mg dry cells/ml. Cells were harvested by centrifugation after the addition of chloramphenicol (50 fig/m\), washed three times with 20 mM TrisHC1 buffer, pH 7.6, and kept frozen at -20° until use. Preparation of Cell-free Extract — The washed cells were suspended in an equal volume of 20 mM Tris-HCl buffer, pH 7.6, containing 10 mM MgCl2 and disrupted with a sonicator (Tominaga UR 150P) at 20 kHz, 74 watts for 4.5 min. The sonicated preparation was centrifuged at 8,000 xg for 10 min. The supernatant was again centrifuged at 25,000 x g for 30 min. The resulting supernatant, containing 45—60 mg protein per ml, was employed as crude extract. The activities of succinate dehydrogenase [EC 1.3.99.1] and Dlactate dehydrogenase (NAD+-independent) in the resulting precipitate amounted to less than 7% of those in the supernatant. Because of this, only the enzymic activities in the supernatant were measured. Reagents — NAD+, NADH, NADP+, 3acetylpyridine adenine dinucleotide (APAD+), and Co A were purchased from Sigma. Assays of Enzyme Activities—Assay systems for each enzyme are listed in Table I. All measurements were performed at 25° with a Hitachi double-beam spectrophotometer, model 356, or Gilford spectrophotometer, model 2400. The rates of enzymic reactions were obtained from the differences in the rates of increase or decrease of absorbance in the presence and absence of the substrates. Molecular extinction coefficients of 6.2(5), 9.1(5), and l.OmM-'-cm" 1 {10) were employed for NAD(P)H (at 340 nm), APADH (at 365 nm), and ferricyanide (at 420 nm), respectively. The rates were found to be / . Biochem.

EFFECT OF NITRATE REDUCTION ON ENZYME LEVELS IN E. coli

309

TABLE I. Assay systems for enzyme activities. Enzyme

Substrate

Buffer and other component

Aconitate hydratase

Citrate 10 mM

Tris-HCl, pH+ 7.6, 100 mM, MgCl2 10 mM, and NADP 0.3 mM

NADPH at 340 nm

Isocitrate dehydrogenase

Isocitrate 10 mM

Tris-HCl, pH 7.6, 100 mM, MgCl2 10 mM, and NADP+ 0.3 mM

NADPH at 340 nm

2-Oxoglutarate dehydrogenase

2-Oxoglutarate 0.9 mM

Tris-HCl, pH 7.6, 100 mM, KCN 10 mM, CoA 0.07 mM, cysteine 3 mM, and APAD+ 0.15 mM

APADH at 365 nm

Succinate dehydrogenase

Succinate 10 mM

Tris-HCl, pH 7.6, 100 mM, MgCl2 10 mM, and K3Fe(CN)6 1 mM

Fe(CN)63" at 420 nm

Malate dehydrogenase

Oxaloacetate 3mM

Tris-HCl, pH 9.0, 100 mM, KCN 10 mM, and NADH 0.15 mM

NADH at 340 nm

Pyruvate dehydrogenase

Pyruvate 10 mM

Tris-HCl, pH 7.6, 100 mM, KCN 10 mM, CoA 0.1 mM, cysteine 3mM, TPP 0.2 mM, MgClj 10 mM, and NAD+ 1 mM

NADH at 340 nm

Malic enzyme

Malate 2mM

Tris-HCl, pH 7.6, 100 mM, MgCl2 10 mM, KC1 10 mM, and NADP+ 0.3 mM

NADPH at 340 nm

Glutamate dehydrogenase

Glutamate 10 mM

Tris-HCl, pH+ 7.6, 100 mM, MgCl2 10 mM, and NADP 0.3 mM

NADPH at 340 nm

Glucose-6-phosphate dehydrogenase

Glucose 6-phosphate 2mM

Tris-HCl, pH+ 7.6, 100 mM, MgCl2 10 mM, and NADP 0.3 mM

NADPH at 340 nm

Alcohol dehydrogenase

Acetaldehyde 40 mM

Lactate dehydrogenase (NAD+-dependent)

Pyruvate 10 mM

Na, K phosphate buffer, pH8.2, 100 mM, KCN 10 mM, and NADH 0.15 mM Na, K phosphate buffer, pH 6.4, 100 mM, KCN 10 mM, and NADH 0.15 mM

NADH at 340 nm NADH at 340 nm

D-Lactate dehydrogenase (NAD+-independent)

D-Lactate 10 mM

Tris-HCl, pH 7.6, 100 mM, KCN 10 mM, MgCl2 10 mM, and K3Fe(CN)6 1 mM

Fe(CN)63" at 420 nm

proportional to the amount of extract below the following values: 120 //M/min for aconitate hydratase [EC 4. 2.1.3], 600 /iM/min for isocitrate dehydrogenase [EC 1.1.1.42], 55 /M/min for 2-oxoglutarate dehydrogenase [EC 1.2.4.2], 30 /*M/min for succinate dehydrogenase, 500 pM/ min for malate dehydrogenase [EC 1.1.1.37], 110 /*M/min for pyruvate dehydrogenase [EC 1.2.4.1], 20 ^M/min for malic enzyme [EC 1.1.1.40], 40 fiM/min for glutamate dehydrogenase [EC 1.4.1.3], 260 //M/min for glucose6-phosphate dehydrogenase [EC 1.1.1.49], 8 /^M/min for alcohol dehydrogenase [EC 1.1.1.1], 450 fiM/min for lactate dehydrogenase (NADMependent) [EC 1.1.1.28], and Vol. 78, No. 2, 1975

Measurement

100 /iM/min for D - lactate dehydrogenase (NAD+-independent). All assays were carried out with amounts of the extracts containing activities under these limits. Specific activities are given in nmoles/min/mg protein. In the assay of aconitate hydratase, isocitrate formation from citrate was followed by NADP+ reduction coupled to isocitrate dehydrogenation (11). Isocitrate dehydrogenase was not added to the reaction mixture, as the dehydrogenase activity was sufficiently high in all the extracts. For each extract, the activities of the individual enzymes were measured 2—5 times and mean values were obtained. The deter-

310

I. YAMAMOTO and M. ISHIMOTO

minations were repeated for 3—4 different preparations of cells grown aerobically and anaerobically in the presence and absence of nitrate and S.E. as well as mean values were obtained. For inhibition experiments with nitrate and nitrite, extracts from cells grown under various conditions were employed. Other Assays—Protein was determined by the method of Lowry et al. {12). Bovine serum albumin was used as a standard. Nitrite was determined by a diazo-coupling method (73). Glucose was assayed by the method of Somogyi-Nelson (14) after elimination of nitrite by treatment with sulfamic acid (75).

|0.50

RESULTS 24 Changes of Enzyme Activities during Cul4 8 12 16 20 INCUBATION TIME ( hr ) tivation—The activities of four enzymes, isocitrate dehydrogenase, aconitate hydratase, Fig. 1. Cell growth, glucose consumption, nitrite glucose-6-phosphate dehydrogenase, and glu- accumulation, and enzyme levels during anaerobic tamate dehydrogenase were followed in cell- cultivation in the presence of nitrate. Experimental free extracts from cells grown anaerobically details are given in "EXPERIMENTAL PROCEin the presence of nitrate (Fig. 1). At the DURE." Extracts for enzyme assay were obtained same time, the amounts of glucose and nitrite from cells harvested after 8, 12, 16, and 23.5 hr as well as the amount of cells were measured. (incubation in different flasks). A, x , Absorbance A phase of rapid cell growth during incuba- at 660nm; D, glucose concentration, and • , nitrite tion for 4—8 hr was succeeded by a stationary concentration. B, Activities of enzymes. O, Glutamate dehydrogenase; • , isocitrate dehydrogenase; phase which began after incubation for 12 hr. A, glucose-6-phosphate dehydrogenase; and A, acoSome glucose remained and the amount of nitate hydratase. nitrite was still increasing at this time. Maxima of specific activities of isocitrate dehydrogenase, aconitate hydratase, and glucose- tions of incubation were 6 and 8 hr in most 6-phosphate dehydrogenase were found between cases for aerobic and anaerobic cultivation, re8 and 12 hr incubation, while that of glu- spectively. tamate dehydrogenase was observed at 12 hr; The rates of cell growth in these cultivai.e., the maxima came at the end of the ex- tions were determined by measuring the abponential phase or at the beginning of the sorbance of the cultures every 30 min. The stationary phase of growth. In further ex- rate in the initial phase was found to be about periments for the comparison of enzyme ac- 1.5 fold higher in the anaerobic culture with tivities, cells were always harvested from 8 hr nitrate and in the aerobic culture than in the cultures with absorbance of 0.4—0.5 at 660 nm. anaerobic culture without nitrate. The ratios As a similar level of cell growth was attained of the amounts of cells grown to the amount under anaerobic conditions in the absence of of glucose consumed in the initial phase of nitrate, cultures with the same absorbance growth were, for example, 34, 120, and 21 g were also employed in this case. Although a dry cells/mole of glucose for cells grown in higher growth level was achieved under aerobic anaerobic culture with nitrate, in aerobic culconditions, cells were obtained from the cul- ture, and in anaerobic culture without nitrate, tures with the same absorbance. The dura- respectively.

/ . Biochem.

Vol. 78. N

p

M T)

TABLE II. Activities of enzymes in extracts from cells grown aerobically or anaerobically in the presence and absence of nitrate. Ex per imental details are given in the text. Figures in parentheses show the number of preparations used in the measurement.

to to en

Enzyme Aconitate hydratase Isocitrate dehydrogenase 2-0xoglutarate dehydrogenase Succinate dehydrogenase Malate dehydrogenase Pyruvate dehydrogenase Malic enzyme Glutamate dehydrogenase Glucose-6phosphate dehydrogenase Alcohol dehydrogenase Lactate dehydrogenase (NAD+-dependent) D-Lactate dehydrogenase (NAD+-independent)

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( b )

( c )

( a )

( c )

( d )

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( b )

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HK 1 5 . 4 " ( 4 )

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0.60 ( 3 )

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4.40 ( 4 )

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115

+ 11.7 ( 4 )

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0. 2

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0.28 ( 4 )

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0. 9

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574

+ 95.7 ( 3 )

403

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2.56 ( 3 )

71. 4 +

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3.5

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1.27 ( 4 )

7. 3 +

0.45 ( 4 )

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0.60 ( 4 )

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23. 5 + "

4.26 ( 4 )

16. 5 + "

0.74 ( 4 )

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0. 7

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1. 3

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0. 9

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1. 1

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5. 8

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135

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26

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4. 3 +

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0.39 ( 4 )

0.84 ( 4 )

( 3 )

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5. 4 +

7. 5 + ( 2 )

8..6 +•

0.63 ( 4 )

12. 4 + "

1.30

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26. 4 + "

5.16 ( 4 )

110

+ 14.7 ( 4 )

2. 6 +

0.04 ( 3 )

+ "

7.82 ( 3 )

10. 8 +

0.76 ( 4 )

170

100

14 . 3 +

0.87

( 3 ) 201

+ 10.9

0. 7

( 3 )

4 .5 +

0.88 ( 4 )

1. 0

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