Assimilation of citric acid and adipic acid by the blue ...

Assimilation of citric acid and adipic acid by the blue-green alga Anabaena variabilis M. BUTLER^ A N D J. B. CAPINDALE Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 192.64.11.124 on 06/03/13 For personal use only.

Depcrrttnent of Chemistry, Utliversity of Wnterloo, Wnrerloo, Otlmrio

Accepted May 7, 1975 BUTLER.M., and J. B. C A P I N D A L E1975. . Assimilation of citric acid and adipic acid by the blue-green alga At~ahrretlnvnriohilis. Can. J . Microbiol. 21: 1372-1378. The assimilation of [1.6-I4C] citric acid and [1,6-I4C] adipic acid by the blue-green alga Atlrrbrrrtln vnrirrhilis was studied in the dark and in the light. Citric acid was assimilated in the dark and in the light but adipic acid showed only limited assimilation in the dark. In the light the assimilation of adipic acid did not enhance the growth of the alga at a concentration of 2.85 x M. Growth was inhibited at adipic acid concentrations greater than 10-3 M. Analysis of the products of adipic acid metabolism showed the presence of aspartic acid, glutamic acid. leucine, proline, and threonine amongst other unidentified compounds. A mechanism of p-oxidation is proposed. BUTLER,M., et J. B. C A P I N D A L E1975. . Assimilation of citric acid and adipic acid by the blue-green alga Atltrhaetzrr \~crrirrhilis.Can. J . Microbiol. 21: 1372-1378. L'assimilation de 1 1,6-I4C] acide citrique et de [1,6-I4C] acide adipique par I'algue bleu-vert Atrrrhnetlcr vcrricrhilis a kt6 etudiee a I'obscurite et i la lumiere. L'acide citrique est assimile et 5 I'obscurite et B la lumiere, mais I'acide adipique ne montre qu'une assimilation limitee h I'obscurite. A la lumiere, I'assimilation de I'acide adipique n'augmente pas la croissance de I'algue h la concentration de 2.85 x 1 0 - W . La croissance est inhibee aux concentrations plus elevees que M. L'anzllyse des produits du metabolisme de I'acide adipique montre, en plus de composes non identifies. la pltsence d'acide aspal-tique, d'acide glutamique, d e leucine, de proline et de threonine. On propose Lrn micanisme d e p-oxydation. [Traduit par le journal]

Introduction Despite the classification of blue-green algae as obligate autotrophs much has been published on the assimilation of organic compounds by these organisms (for review see ref. 15). The presence of these compounds does not, however, increase the growth rate of the blue-green algae in the light (9, 11, 14, 16, 17). Heterotrophic growth has been shown in the dark and in dim light only in certain cases (2, 6, 17). Incorporation of radioactively labelled organic substrates into cellular components has been shown (11, 12, 17). In the case of incorporation of [U-14C] glucose and [2-14C] acetate 18% to 32% of the cellular dry weight of Anabaena uariabilis was derived from labelled substrates (11, 12). The metabolism of these organic substrates has been related t o a n incomplete tricarboxylic acid (TCA) cycle in Anabaena variabilis (I 3). 'Received February 4, 1975. 'Present address: Department of Chemistry a n d Biology, Manchester Polytechnic, Chester Street, Manchester' M I 5GD, Great Britain.

In the present work the incorporation of radioactively labelled citric acid and adipic acid into Anabaeila uariabilis was studied both under dark and light growth conditions. Citric acid was originally added to an algal growth medium as a chelating agent to facilitate the uptake of heavy metal ions (4, 9). The metabolism of adipic acid was compared with its utilization by Pseudomonas fluorescens in which four inducible catabolic enzymes have been shown following growth in media containing dicarboxylic acids (7).

Materials and Methods An inoculum of Anabnena vorinbilis was obtained from the Botany Department, Indiana University, U.S.A. Specifically labelled radioactive compounds [1,6-'4Cl citric acid and [1,6-14C] adipic acid were purchased from New England, 575 Albany St., Boston, Massachusetts, U.S.A. 1,4-bis-[2-(5-phenyloxazolyl]-benzene (POPOP) and 2,5-diphenyloxazole (PPO) were purchased from Amersham/Searle Corp., 2000 Nuclear Drive, Des Plaines, Illinois, U.S.A. Medirrtn

The mineral medium of Hughes et nl. (8) was used without added nitrate. When adipic acid incorporation

BUTLER AND CAPINDALE: ACID ASSIMILATION BY ANABAEUA

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was studied the citric acid normally present was replaced by 0.0042 gllitre adipic acid and the ferric citrate by 0.0037 g/Iitre ferric chloride. This represented a mole-to-mole substitution of each. Gro)vt/~ Growth was maintained in sterilized medium (200 ml to 1.5 litre) at 27'C with constant aeration. Illumination was provided by two banks of fluorescent lights maintained on a 16-h light - 8-h dark cycle. The intensity of the light was 600 ft-c at the surface of each flask. Dark growth experiments were performed by covering the flasks with black photographic paper. The absorbance of the algal culture at 600nm was used to measure growth (5). Chr.omnto~~.ap/iy Descending chromatography was performed on 25.4cm x 20.3 cm Whatman N o 1 paper. The solvent systems used were ti-butano1:aceticacid :water (120: 30: 50 by vol.); ethanol :water :ammonia (180.10.10 by vol.); 11-butanol :pyridine .water (60: 60: 60 by vol.). Electroplioresis This was performed in all glass tanks using 0.1 M (NH,)2C03 buffer, p H 9.0. Power was provided by a Heathkit power-pack model IP-32 Daystrom Ltd. Autoradiograpliy Radioactive spots were identified by placing the chromatograms in contact with X-ray film (Kodak RB-2) from 4 to 10 days. Radiocliro~~iatogranz Scanning T h ~ swas performed with a Packard model 7200 radiochromatogram scanner. Perrnatlgat~ateOxidation Each sample was refluxed with 2.0 ml potassium permanganate (7.5 mM) at pH 2.0 for 30 min. The reduction of absorbance at 525 nm indicated the overall organic content. Scintillatiotz Corrtztif~g This was performed with a Packard scintillation counter. The scintillation fluid contained 42 ml Spectrofluor in toluene (700 mi) and ethanol (300 ml). The Spectrofluor was a solution of 1.25 g POPOP and 100 g PPO in 1 litre of toluene.

Results Uptake of Citric Acid as Shown by the Pertnanganate Oxidation Method The variation in organic content of the medium of an algal culture grown in the light over a period of 160 h is shown in Fig. 1. The initial inoculation of algae was 0.5 mg dry weight per millilitre of medium. Samples (10 ml) were taken at time intervals from the 1.5-litre culture. After removal of algae by centrifugation each sample was treated with potassium permanganate to determine the organic content of the medium. The uptake of citric acid is shown by a decrease in the oxidizu

04 -

0 20 40 60

80 100 120 140 160 H

FIG.1. Variation of the organic content of the medium of an algal culture with time a s determined by the permanganate oxidation method. After inoculation 10-ml samples were taken at time intervals from a 1.5-litre culture. After removal of algae by centrifugation each sample was refluxed with 2.0 ml potassium permanganate (7.5 mM) at pH 2.0 for 30 min. The consumption of permanganate by each sample was measured by the reduction in absorbance at 525 nm.

able content of the medium up to 20 h. This is followed by an increase which is continued until the final reading at 160 h. The increase is attributed t o extracellular release of various organic materials by the algae. The uptake of citrate by the algae was confirmed by the use of 14C-citric acid. Uptake o f (I$-14C] Citric Acid and [1,6-14C] Adipic Acid The change in radioactive content of the medium for algal growth in the presence of [1,6-14C] citric acid and [1,6-14C] adipic acid both in the dark and in the light is shown in Fig. 2. The initial inoculation in all cultures was 0.5 mg dry weight of algae per millilitre of medium. Samples (10ml) were taken at time intervals and after removal of the algae the radioactive content of the medium was determined by scintillation counting. Figure 2a shows that in the light the incorporation of citric acid continued for 50 h at a rate of 5.5 x 10-l3 rnol citratellitre of medium per hour. In the dark the rate of uptake of citric acid was higher for the first 10 h and then an increase in the radioactive content of the medium was shown. This increase is probably due to radioactive incorporation from citric acid t o compounds released extracellularly. The uptake of [1,6-14C] adipic acid is shown in Fig. 2b. The rate of uptake of adipic acid from the medium was 2.3 x mol adipatellitre of


CAN. J. MICROBIOL. VOL.

FIG.2. Assimilation of [1,6-14C]citric acid and [1,6-14C] adipic acid by an algal culture grown in the dark and in the light. (a)Citric acid. (b) Adipic acid. After inoculation 10-ml samples were taken at time intervals

from a 500-ml culture containing either 14C-citricacid (0.32 mCi/mMol) or 14C-adipicacid (6.00 mCi/mMol). After removal of algae by centrifugation each sample was assayed for radioactivity by liquid scintillation counting. (a) shows assimilation in the dark and (0) shows assimilation in the light. medium per hour for the first 10 h of growth. This represented a much higher molar uptake for adipic acid than for citric acid. For growth in the dark no adipic acid was assimilated by the algae after the first 4 h after inoculation. Identification of the Products of the Metabolism of Adipic Acid Algal cells were isolated after growth in a medium containing [1,6-14C] adipic acid (2.67 mCi/prnol). Aliquots of a hot ethanol extract were spotted on to paper for electrophoresis in one dimension followed by chromatography in the second dimension. The solvent system used for chromatography was butanol :acetic acid :water. Electrophoresis was conducted at 400 V for 1 h. Radioactive spots were identified by autoradiography. The hot ethanol extract after 2 h and 4 h of algal growth contained radioactive adipic acid and an unidentified radioactive compound. Further aliquots of the extract were chromatographed in other solvents. The chromatographic properties of the unidentified compound are given in Table 1. Treatment of the compound with bromine did not affect the chromatographic properties. This indicated that the compound was not unsaturated. The hot ethanol extract of the alga after 20 h of growth in [14C] adipic acid contained a variety of radioactively labelled compounds. Cochromatography with standard amino acids enabled identification of some of the radioactive

spots which were ninhydrin-positive. The following amino acids were positively identified: aspartic acid, threonine, glutamic acid, proline, and leucine. These corresponded to spots 1 t o 5 res~ectivelvof Table 2. S ~ o 8t was identified a s h i p i c acid and spot 9 'corresponded t o the unidentified compound found t o be radioactively labelled after 2 h. Spot 6 was pigmented and thought t o be chlorophyll. Spots 7 and 10 were unidentified. When a strip of the chromatographic paper was passed through a radiochromatogram scanner the radioactive content of three of the amino acid spots were compared before and after ninhydrin treatment. For threonine, proline, and leucine the loss in radioactivity varied from 30 to 5 0 x after ninhydrin treatment. Since ninhydrin liberates the carboxyl group of an amino acid these percentages correspond t o the proportion of 14C contained in the carboxyl group of the amino acid molecules. Lipids were extracted from the algal cells after 20 h incubation with [14C] adipic acid by shaking with chloroform :methanol (2 : 1 v/v) at room temperature for 3 h (3). Water-soluble compounds were removed from the concentrated extract by shaking with 0.2 volumes of water. The concentrated sample of the extract was chromatographed on a silica plate in chloroform :methanol :acetic acid :water (170: 30: 20: 7 by vol.). Lipid spots were shown by placing the dried silica plate in iodine vapor. Out of the four spots shown by this method two werc -

-

BUTLER AND CAPINDALE: ACID ASSIMILATION BY ANABAENA

TABLE I Chromatographic and electrophoretic characteristics of adipic acid, and a radioactively labelled compound found after 2 h to 4 h algal growth in [14C] adipic acid

Rf values Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 192.64.11.124 on 06/03/13 For personal use only.

Solvent system n-Butanol :acetic acid :water (120:30:50) Ethanol :water :ammonia (180: 10: 10) n-Butanol :pyridine :water (60 :60 :60) Distance travelled towards anode by electrophoresis

Unknown

Adipic acid

0.92

0.85

0.76

0.14

0.19

0.61

3.6-cm

7.9-cm

NOTE:A concentraled hot ethanol ehtract was made o f the algae after 2 h and 4 h in a medium containing [1.6-'4Cl adipic acid. Descending paper chromatography was performed in various solvent systems. T h e chromatograms were developed by autoradiography. Conditions of electrophoresis: 100 V for 90 min using 0.1 M (NH&C O , buffer, p H 8.7.

TABLE 2 Chromatographic and electrophoretic characteristics of radioactively labelled compounds formed after 20 h of algal growth in [14C] adipic acid Spot No. I

2 3

4 5 6 7 8 9 10

R f.

Distance travelled towards anode, cm

0.26 0.36 0.40 0.46 0.71 0.83 0.87 0.89 0.92 0.96

6.0 zero 3.8 zero zero 4.3 3.0 5.8 3.8 zero

Ninhydrin sensitivity

+ + + + + -

-

-

Identification by cochromatography asp thr glu Pro leu

-

Adipic acid

-

Pigment probably chlorophyll

NOTE:A concentrated hot ethanol extract was made o f t h e algae after 20 h ofgrowth in a medium containing adipic acid. Two-dimensional electro~horesis-chromatography enabled separation of the cornponenls o f the extracl. Electrophoresis was conducted in the first direction a t 400 V for I h using 0.1 M (NH4)2COJ buffer. pH 9.0. Descending chromatography was performed in an ?r-butanol:aceric acid:water (120:30:30) solvent mixture. The chromatogram was developed by autoradiography and was also sprayed with ninhydrin. .

.

*

radioactive and had Rf values of 0.63 and 0.87. These two iodine-sensitive spots were in similar positions to the diglyceride spots identified by Nichols et at. (10) from a similar lipid extraction of Anubuenrr z>rrrir~bilis. Also a green pigment a t an R, value of 0.90 was radioactive. This was thought to be chlorophyll.

sufficient stirring from air bubbling through the medium. It was shown over a series of 10 growth experiments that a low concentration of adipic acid (2.55 x M) had no effect on the growth of the alga. Inhibition ofgrowth was, however, shown by concentrations greater M adipic acid. than

EfSect oj'Adipic Acid on the Growtll of Anabnena variabilis Growth experiments were conducted in 20-ml cultures in test tubes, where a homogeneous suspension of the alga was maintained by

Discussion The uptake of citric acid was studied by two different methods. The permanganate oxidation method measures the overall organic content


1376

CAN. J. MICROBIOL. VOL. 21, 1975

of the medium. This method showed that after rapid uptake of citric acid, extracellular release of organic content by the alga raised the organic content of the medium (Fig. 1). This extracellular release of organic material which rose after 20 h may have begun at the point of inoculation. The function of citric acid in the algal medium is to chelate metal ions (4, 9). It is possible that the chelating function is continued by the organic material released by the alga after the rapid depletion of citric acid. The experiments using [14C] citric acid showed that the compound was entirely assimilated by the alga in the light and that none of the radioactivity passed onto the material released extracellularly (Fig. 2a). Assimilation of citric acid in the dark occurred at an initially more rapid rate but after 10 h of growth the radioactive content of the medium increased. This increase was due either to the incorporation of radioactivity into compounds released extracellularly or to lysis of the algal cells. In the case of [I4C] adipic acid uptake was shown by the alga grown in the light but only limited uptake was shown in the dark. In a similar set of dark and light experiments using Anabaetla cariabilis, Pearce and Carr (I I) found that the uptake of [14C] acetate was the same in the 1st h for light and dark growth. However, after 1 h no further uptake of acetate was shown in the dark. A tentative explanation for these dark effects may be related to photophosphorylation. Acetate would normally be converted to acetyl CoA in the cell; a reaction which requires adenosine 5'triphosphate (ATP). Acetate uptake in the dark may cease once the photosynthetically generated ATP is used. However, citrate does not require any priming reaction and so may be metabolized without ATP. Similarly it might be suggested that one of the initial reactions of adipic acid in the cell involves utilization of ATP and once this is depleted no further assimilation of adipic acid can take place. Smith et al. (16) studied the assimilation of [14C] acetate by Anacystis nidulans and found radioactive incorporation into leucine, glutamic acid, proline, and arginine. Leucine is synthesized from acetyl CoA and the other three form the glutamic acid group of amino acids which are all derived from a-ketoglutarate. Assimilation of [14C] pyruvate was found by

Smith et al. (16) to bring about radioactive incorporation into valine, alanine, leucine, and the glutamic acid group of amino acids. Pearce and Carr (1 1) have shown an incomplete TCA cycle in Anabaena uariabilis by the absence of the enzymes, a-ketoglutarate dehydrogenase, and succinyl CoA synthetase. This explains the inability of the alga to incorporate radioactivity from [14C] acetate into the aspartic acid group of amino acids which are derived from oxaloacetate and include isoleucine, threonine, lysine, and methionine. In the present work the amino acids leucine, glutamic acid, proline, aspartic acid, and threonine were found to have incorporated radioactivity from [l ,6-14C]adipic acid. The suggested metabolism of adipic acid is initially by cleavage into acetate and succinate possibly by the process of 0-oxidation similar to the mechanism for fatty acid catabolism. Such a cleavage of specifically labelled [1,6-'~Cladipic acid would lead to acetate and succinate molecules, each containing a 14C atom. This would suggest that one [14C] carboxyl group of adipic acid gave rise to [14C] acetyl CoA which led to radioactive incorporation into leucine, glutamic acid, and proline and the second [14C] carboxyl group which led to radioactive incorporation into aspartic acid and threonine through [14C] succinate. Succinate could give rise to oxaloacetate and hence aspartic acid and threonine through one branch of the incomplete TCA cycle. Ninhydrin spraying of the three radioactive amino acids threonine, proline, and leucine reduced the radioactivity of each by 30 to 50% This indicates the presence of at least twc 14c-atoms in each amino acid molecule, onc of which must be the a-carboxyl carbon. Thi: is in fact compatible with the labelling patterr expected for the generally accepted biosynthetic pathways of each of these amino acids fron the specifically labelled precursors acetate an( succinate which would be formed by cleavage o [1,6-14C] adipic acid. The probable pathway fo radioactive incorporation into the amino acid is shown in Fig. 3. The radioactive incorporatiol into lipids could arise from '4C-labelled acetate Hoare et al. (5) found that assimilation o [14C] acetate into four species of blue-greel algae gave high radioactive incorporation intl the lipid fraction. The radioactivity of chlorc

BUTLER AND CAPINDALE: ACID ASSIMILATION BY ANABAENA

*

=

'4 C

atom

( I ) Cleavage of adipic acid hy P-oxidation '~0.0~ *CO.SCOA *CO.SCOA *CO.SCOA

1 CH2 I CH2


I

I

I CH2 I

+ I

CH2

CH2

CH

II

CH

- 1

CH2

I

I CH2 I

- 1

I

I

CH2

CH.OH

\ I

I

CO..~OA

C=O

- 1

CH2

I

I

*CO.SCOA CH3

I

CH2

CH2

I

*CO.SCOA

CH2

I

12) Convel.\lon of \~lccinylCoA to oxaloacet:rte CO.SCoA *C02*$02*C02-

*CO.SCoA

I

CH3

C&

I

C=O

I

CH3

I

CH3\*

*C=O --+

I I

HO-C-CH3

co2-

__f

/CH3 C-OH

I

CH.OH

I

co2-

CH,\*

+

,CH3 CH

I

C=O

1

CO,

*CO.SCoA

I

CH3

CH3\*

CH3 CH

I

' -OzC-C-OH

+

I

FH2

FIG.3. Possible pathway of incol-polation ol' ~ ~ d i o ; ~ c l i vinto i t y three amino i~cidst'l.om [ 1 .6-I4CIatlipic acid.


1378

CAN. J . MICROBIOL. VOL. 21, 1975

phyll is explained by incorporation from succinate. The incorporation and dissimilation of adipic acid has been shown in bacteria (1 7). Chapman and Diggleby (1) showed that acetyl CoA and succinyl CoA resulted from the catabolism of ad,pic acid i n two bacterial H~~~and Stanier (7) showed that four enzymes are induced in Pseudoi??onas fluorescens by growth in a medium containing adipic acid. Catabolism of adipic acid followed a pathway mechanistically to the P-Oxidation Of acids. A similar catabolic pathway for adipic acid seems highly probable in Anabaena variabilis in view of our evidence. 1, 2.

3. 4,

5.

6.

7. HOET,P. P., and R. Y. STANIER. 1970. Dissimilation of higher dicarboxylic acids by Pserrdomoncrs j u o rescetls. Eur. J . Biochem. 13: 65-70. 8, HUGHES, E , 0,, P, R, GORHAM, and A, Z E H N D E R . 3 1958. Toxicity of a unialgal culture of Microcystis oerrrginoscr. Can. J. Microbiol. 4: 225-236. 9. KRATZ,W. A,, and J. MYERS.1955. Nutrition and growth of several blue-green algae. Am. J . Bot. 42: 282-287. 10. NICHOLS, B. W., R. V. HARRIS, and A. T . JAMES. 1965. The lipid metabolism of blue-green algae. Biochem. Biophys. Res. Commun.20: 256-262. I I. PEARCE, J . , and N . G. CARR.1967. The metabolism of acetate by the blue-green algae Atzcrbt~enrr~~oricrbilis and Anncystjs nidr~,ans,J , Gen, Microbial, 49: 301313. 12. PEARCE. J . , and N. G. CARR.1969. The incorporation and metabolism of glucose by Atltrbtienn twrinbilis. J. Gen. Microbiol. 54: 451-462. and N.G. 1969. The 13. PEARcE J . , C. K. C H A p ~ , P, & ~J ,., and R , G, D I G G L ~ ~1967, y , Dicar. incomplete tricarboxylic acid cycle in the blue-green boxylic acid catabolism by bacteria. Biochem. J . 103: J . Gen. Microbiol. 55: algae. Annhnenn ~~ar~inhilis. 7C. 371 -378. FAY.P. 1965. Heterotrophy and nitrogen fixation in 14. PINTNER, I . J . . and L. PROVASOLI. 1958. Artificial CllloroRloeo,fii~yttYC'I1ii, J , Gen, Microbial, 39: cultivation of a red pigmented marine blue-gl-een alga, FOLCH,J . . M. LEES,and G. H. SLOANE-STANLEY. Pi~ortnidirrmpersicinirim. J . Gen. Microbiol. 18: 1901957, A simplemethod for the isolationof total lipides 197. from animal tissues. J. Biol. Chem. 226: 497-509. 15. S M I T H ,A. J. 1973. Synthesis of metabolic interGERLOFF. G, C,. G. P, FITZGERALD. and F. SKOOG, mediates. It1 The biology of blue-green algae. Edited 1950. The mineral nutrition of Coccochloris penioby N. G. Carrand B. A. Whitton. Blackwell Scientific c y s ~ i sAm. . J . Bot. 37: 835-840. Publications, Oxford, London, Edinburgh, and MelHOARE,D. S., S. L. HOARE, and R. B. MOORE.1967. bourne. pp. 1-38. The photoassimilation of organic compo~lndsby au16. S M I T HJ.. J . , J . LONDON, and R. Y.STANIER. 1967. blue-green algae. J . Gen. Microbial, 49: The biochemical basis of obligate a~~totrophy in blue351-370. green algae and thiobacilli. J . Bacteriol. 94: 972-983. and HOARE,D. s.. L. 0 . I N G R A M . E. L. THURSTON. 17. V A N B A A L E N . C.. D. S. HO*REy and E. BRANDT. R. W A L K U P1971. . Dark heterotrophic growth of an 1971. Heterotrophic growth of blue-green algae in dim endophytic blue-green alga. Arch. Mikrobiol. 78: light. J. Bacteriol. 105: 685-689. 310-321.