Biochimico et Biophysics Acta, 720 ( 1982) 3 11-3 19

Elsevier Biomedical Press

311

BBA 11028

r3C-NMR STUDY OF CO, -FIXATION DURING THE HETEROTROPHIC GROWTH IN CHLOROBIUM THIOSULFA TOPHZLUM

TOOMAS PAALME, ABIRA OLIVSON and RAW0 VILU*

Institute of Chemical Physics and Biophysics of Estonian Academy of Sciences, Tallinn 200 001 (USSR)

(Received September Ist, 1981)

(Revised manuscript received January 27th, 1982)

Key words: “C-NMR, CO, fixation, Heterotrophic growth: (Chl. thiosulfatophilum)

The functioning of the biosynthetic pathways of the amino acids alanine, glycine, aspartic acid, glutamic acid and tyrosine, and of nucleosides in the photosynthetic bacterium Chlorobium thiosulfatophilum during heterotrophic growth on 13C02 and unlabelled acetate was investigated using t3C-NMR as the method for determination of the labelling patterns of the separated substances. On the basis of the analysis of the multiplet structure of the spectra of the tightly-coupled systems, the conclusion was drawn that the Calvin cycle does not function in the experimental conditions used. The labelling pattern of the glutamic acid indicated that about 30% of the amino acid molectdes were synthesized through the reactions of the reductive carboxylic acid cycle, the remaining 70% being derived from oxaloacetate and exogenous acetate through the reactions of the Rrebs cycle. Labelling patterns of the nucleosides were in agreement with their known biosynthetic pathways.

Introduction

In recent years carbon- 13 has been increasingly used as a tracer in the biosynthetic studies of

secondary metabolism, localization of the 13C in the end-products of the metabolism being de- termined by examination of their proton noise-de-

coupled 13C-NMR spectra [l-3]. In the studies of

primary metabolism, only a few attempts have been made to use 13C-NMR [4]. The amounts of the intermediates of the metabolic pathways needed for the determination of the labelling pat- tern by 13C-NMR are too great to be obtained easily. However, it should be remembered that several ‘end-products’ of the metabolism of low- molecular-weight compounds which accumulate in the proteins, nucleic acids and polysaccharides in

*To whom correspondence should be addressed.

great amounts, i.e., certain amino acids, nucleo- tides and sugars, should have labelling patterns in

common with metabolic intermediates, important from the regulatory point of view: pyruvate, oxaloacetate, a-ketoglutarate, 3-phosphoglycerate,

ribose 5-phosphate, etc. In this work the labelling pattern of alanine, glycine, glutamate, aspartate, tyrosine, glucose and uridine was determined and was used to elucidate the network of carbon assi- milation pathways in Chlorobium thiosulfatophilum during heterotrophic growth on acetate and 13C0,. Preliminary results of the study have been pub- lished in Ref 5.

C. thiosulfatophilum is a strictly anaerobic bacterium and depends on a hydrogen donor, light and CO2 for its growth [6]. Ferredoxin-dependent carboxylation reactions catalyzed by pyruvate and cY-ketoglutarate synthases [7] have been shown to take place in the bacteria. These reactions, to- gether with the known carboxylic acid cycle reac-

0167-4889/82/0000-Oooo/%O2.7S 0 1982 Elsevier Biomedical Press

312

tions, were postulated to form the reductive carboxylic acid cycle Scheme I ribonucleosides [8]. However, up to now there has been no unequiv- ocal evidence of the functioning of the reductive carboxylic acid in the cells of C. thiosulfatophilum.

Materials and Methods

Chlorobium limicola f. thiosulfatophilum strain lc was kindly provided by Prof. E.N. Kontratieva (Moscow University). The culture was grown anaerobically on Larsen medium [9] containing 0.01% N a 2 S . 9 H 2 0 , 0.2% Na2S203- 5H20, 0.2% NaHI3CO3 (containing, according to the producer ( V / O "Izotope", USSR) 69% carbon-13) and other minor components with addition of 0.2% sodium

acetate under light at 30°C. Bacterial cells were harvested at the late logarithmic stage by centrifu- gation at 20000 g in a continuous-flow centrifuge and washed with water.

Cell fractionation was carried out by the mod- ified procedure of Grainger [10]. The scheme of fractionation is given in Scheme I.

The protein fraction was washed with acetone and di-ethyl ether to remove trichloroacetic acid and was dried in the air. 200 mg protein was hydrolysed in 10 ml 6 N HCI at 110°C for 24h. The hydrolyzate was evaporated to dryness in vacuum and the syrupy residue was dissolved in 5 ml water and filtered to remove insoluble humin. Excess HCI was removed by evaporation twice with water and syrupy residue was dissolved in 3 ml 0.5 N acetic acid.

DNA, RNA, protein, carbohydrates lipids, pigments

+ RNA, DNA, protein

carbohydrate i

washed cells

¢ DNA, protein, carbo-

hydrate

extraction with 10% triehloroacetic acid (90°C, 1 h)

DNA, carbohydrates

precipitation with 2 vol. ethanol (20°C)

polysaccharides I hydrolysis, 1 N H2SO4

(100°C, 2 h)

glucose

extraction with 0.2 N perctdoric acid (0°C, 1 h)

acid soluble

washed three times with ethanol and acetone (-20°C)

¢ lipids, pigments

extraction and hydrolysis with 0.3 N NaOH (37"C, 18 h), after- wards acidified to 0.2 N perehloric acid

¢ ribonucleotides

alkaline phosphatase hydro- lysis (37°C, 18 h)

¢ protein ribonucleotides '

4, DNA (partially hydrolyzed)

Scheme I. The scheme of fractionation of cells used in the work.

313

For the separation of amino acids the scheme proposed by Hirs et al. was used [11]. Aspartate, glutamate and tyrosine were removed from the hydrolyzate on an anion-exchange resin ARA-8p (Olaine, USSR) column 2 × 10 cm operated at flow rate 3 ml/min at 30°C. Aspartate peak was well resolved from the peaks of other amino acids, whereas the glutamate fraction contained also tyrosine. Tyrosine was separated from glutamate using charcoal. The neutral amino acids were sep- arated on a 1.5 × 35 cm column with a cation-ex- change resin Chromex UA-8 (Reanal, Hungary) using several HCI linear gradients (1.5-4 N HC1) and a flow rate of 1 ml/min at 55°C. Fractions of 2 ml were collected. The fractions of individual amino acids were evaporated to dryness in vacuum and the excess HC1 was removed by evaporation twice with water. The final residues were dissolved in 0.8 ml water for measurement of 13C-NMR spectra.

The content of amino acids in the fractions was analysed using the reaction with strongly buffered ninhydrin reagent [12]. Final quantitation of the isolated amino acids was done using an amino acid analyser AAA-881 (Microtech, Czechoslo- vakia).

The fraction of ribonucleotides (about 50 mg) was neutralized with KOH and centrifuged and the supernatant volume was reduced to 10-20 ml by rotary evaporation at 50°C. The nucleotide solution was separated from RNA and desalted on a 4 × 20 cm Sephadex G-10 column using 0.05 M (NH4)2CO 3 at pH 9.0. The fraction of ribonuc- leotides was dissolved in 0.1 N Tris-HCl buffer (pH 10) with 0.01 M MgCI 2. For the preparation of nucleosides, 5 mg alkaline phosphatase (Koch- Light) were added to the solution and it was incubated for 18 h at 37°C. The hydrolyzate was fractionated on the Sephadex G-10 column, de- scribed above, eluted with (NH4)2CO 3 at pH 9.0. The two fractions of uridine + cytidine and adenosine + guanosine obtained were evaporated to dryness and dissolved in 3 rnl 0.25 M NH4C1 solution. Uridine, cytidine and guanosine, adeno- sine were separated on a 1.5 × 30 cm Dowex I-X10 column using 0.25 M NH4CI at pH 9.0 at 30°C. The individual nucleosides were desalted on the Sephadex G-10 column and evaporated to dryness in vacuum. For measurements of the t3C-NMR

spectra nucleosides were dissolved in 0.8 ml di- methylsulphoxide.

~3C-NMR spectra were obtained at room tem- perature on a Bruker WH-90 Fourier Transform spectrometer operating at 22.63 MHz under condi- tions of complete proton noise decoupling and with an internal deuterium field frequency lock. Data were accumulated in a Bruker B-NC-12 com- puter using either 2500 or 1500 Hz bandwidth in 4 K data points. Pulses of 11 /~s pulsewidth (60 °) with a pulse delay of 3.5 s were used. Chemical shifts are given relative to external Me4Si.

The enrichment of the position i in the mole- cules with position j labelled and position k un- labelled, E +j-k, may be calculated using the equa- tion

E + j - k - Dj, Dj, + Sj (1)

where Dji is the total intensity of the doublet, • corresponding to the j th carbon arising due to the spin-spin coupling of the j th carbon with the ith carbon; Sj is the intensity of the singlet of the j th carbon. This equation can be used for the de- termination of the enrichments of the adjacent positions if the resonances of the carbon under study are resolved. In the case of statistically inde- pendent labelling of the carbons under considera- tion, the enrichment determined by this equation is absolute enrichment.

If the absolute enrichment of a position i is known, the enrichment of the position j can be determined by the equation:

E j - -Ki j Mi (2)

where E*~ is the enrichment of carbon j, de- termined by Eqn. 2 using the carbon i as the reference; E/is the known enrichment of carbon i; Mj is the total intensity of the lines corresponding to carbon j (the total areas under the contours of the lines are used throughout this paper); M~ is the total intensity of the lines corresponding to carbon i; K u is the constant which is determined from the natural-abundance J3C-NMR spectrum of the substance and reflects the differences in the intensities of lines of the carbons i and j in the conditions where spectra were measured.

3 1 4

Resu l t s

Several substances were purified according to the scheme of fractionation (Scheme I) of the cells of C. thiosulfatophilum, grown on acetate and 13CO2 (69% of carbon-13), in the amounts more than 0.1 mmol, sufficient for the measurement of their 13C-NMR spectra.

From the protein fraction, the 13C-NMR spec- tra of alanine, glycine, aspartate, glutamate and tyrosine were measured.

Alanine Comparison of the total intensities of the lines

of the carbons in the ~3C-NMR spectrum of the alanine indicates that the level of enrichment of carbon 1 is about 10-times higher than those of carbons 2 and 3 (Fig. 1). Taking into account the

I - - 1 l - - I

J1,2

C~

I I

A L A N I N E

CH 3 H C00H NH 2

C2

L t / / t 410 180 170 60

J3.2

..A, i 03

I 1 20 0

C H E l,d I C A L S H I F T {ppm)

Fig. 1. Proton-decoupled L3C-NMR spectrum of the labelled alanine, separated from the cells of (7. thiosulfatophilum.

assignment of the peaks made in the case of the well-resolved group of lines corresponding to carbon 2 in the spectrum of uniformly labelled alanine [13], the enrichment of the carbons 1 was calculated using Eqn. 1 for the molecules in which carbons 2 were labelled and carbons 3 unlabelled. The calculated level of enrichment, E +2-3 ~- 53%, shows that the carbons 1 in the molecules were derived from exogenous 13 CO2. The enrichment of the carbons 2 in the molecules, labelled at carbons 1 (E~l) , calculated using Eqn. 1, is 4.5%. Assum- ing that the calculated level of enrichment is the true or absolute enrichment of the carbons 2, the enrichment of the carbons 3, determined using in Eqn. 2 the total areas under the contours of the lines of the carbons 2 and 3, turned out to be about 4%. The enrichment pattern of the alanine, which should coincide with that of the pyruvate, is in good agreement with the conclusion that the main bulk of the pyruvate was synthesized from exogenous acetate and 13CO2 through the pyruvate sythase reaction. However, minor enrichment of the carbons 2 and 3 indicates that beside this 'direct' pathway, other reactions of alanine synthe- sis should also take place in the cells of C. thio- sulfatophilum. For the characterization of these additonal reactions it should be noted that E f 3 was 28%.

Glycine The enrichment of the two carbons in glycine,

separated from C. thiosulfatophilurn, was de- terrnJned from the 13C-NMR spectrum by Eqn. 1. It was found that E~ 2= 53% and E ~ - ~ 5%. The 'geometry' of the glycine biosynthesis pathway suggests that the enrichment of the carbons 1 and 2 of the amino acid coincides with that of the carbons 1 and 2 of the 3-phosphoglycerate inter- mediate of the gluconeogenesis pathway. The coincidence of the enrichment of carbons 1 and 2 in the glycine and alanine should be considered therefore as evidence of the functioning of the gluconeogenesis pathway in C. thiosulfataphilum.

Aspartic acid The 13C-NMR spectrum of the labelled aspartic

acid is shown in Fig. 2. The enrichment of posi- tions 1, 3 and 4 in aspartic acid was determined by Eqn. (1): E +2=53%, E~ -4=7.5% and E~ -3 z =

i J4,3~

c41 c,

, //

ASPARTIC ACID

1 2 3 z,

H0OC CH CH 2 C00H NH 2

I L ,

C H E M I CA L

03

C2 J3A~

6'0 4'0

S H I F T (ppm)

Fig. 2. Proton-decoupled 13C-NMR spectrum of the labelled aspartic acid from C thiosulfatophilum.

55%. The enrichment of position 2, calculated by comparison of the total intensities of the lines of carbons 2 and 3 taking K23 ---- 1, is about 6%. The practical coincidence of the enrichment in the aspartic acid with corresponding enrichment in the alanine and exogenous 13CO2 (see TableI and Scheme II) shows that the main bulk of the aspartic acid (and oxalocacetate) was derived directly from pyruvate, probably through the phosphoenol- pyruvate synthase and phosphoenolpyruvate carboxylase reactions [8].

Glutamic acid. The enrichment of carbons 1, 2 and 5 in glutamic

acid, the ~3C-NMR spectrum of which is shown in Fig. 3, determined by Eqn. 1 was: E +2-3 = 55%, Ef 1= 22% and E~ - 4 - 3 = 53%. E ; 4 being 53% the enrichment of the carbon 5, calculated using Eqn. 2 and carbon I as a reference, taking KI5 = 1 (E5"1), was 17%. The enrichment of the carbons 3 and 4, determined by the same equation (Eqn. 2) using carbon 2 as a reference and taking K23 ---- K24 = I was about 9% (see Table I). The main features

315

D

I--

I

l J1'2 /

'.'1

Cs

I

180 L / /

IBo //'

/ / '

GLUTAMIC ACID 5 4 3 2 1

H00C CH:~ CH 2 CH C00H NH 2

C2

J2,1 i C3

60 40 20

C H E M I C A L S H I F T (ppm)

Fig. 3. Proton-decoupled 13C-NMR spectrum of the labelled glutamic acid from C. thiosulfatophilum.

of the described enrichment pattern of the gluta- mate are in good agreement with the observations made by Hoare and Gibson [14], who used in their experiments non-enriched exogenous CO 2 and either (1- J4 C)- or (2-14 C)-labelled acetate.

Glutamate may be synthesized either through the reactions of the reductive carboxylic acid cycle (clockwise direction in Scheme II) or through the reactions of Krebs cycle (anticlockwise direction in Scheme II). In the first case the carbons 1, 2 and 5 of the amino acid are directly derived from exoge- nous 13CO2 (lower marking of label in a-keto- glutarate molecule in Scheme II). In the second case only carbons 1 were derived directly from ~3CO 2 (upper marking of label in a-ketoglutarate), carbons 2 and 3 originating from oxaloacetate unlabelled and carbons 4 and 5 directly from acetate. As seen in Scheme II the absolute enrich- ment of the carbons 1 should be the highest in the molecules and close to that of the exogenous 13CO 2. And indeed, the value of E +2-3= 55% indicates that in those molecules where the carbons 2 were labelled, the carbons 1 were derived from exoge- nous 13CO 2. Taking into account also the total intensities of the lines of the carbons 1 and the other carbons and the value of E~- l __ 22%, which

316

T A B L E I

E N R I C H M E N T O F T H E C A R B O N S IN T H E D I F F E R E N T M E T A B O L I T E S S T U D I E D

These are a r r a n g e d in c o l u m n s a c c o r d i n g to c o n n e c t e d n e s s t h r o u g h the m e t a b o l i c p a t h w a y s , f u n c t i o n i n g a c c o r d i n g to ou r d a t a in C.

thiosulfatophilum. E + i , e n r i c h m e n t o f the p o s i t i o n j was d e t e r m i n e d b y Eqn. 1 f rom the g r o u p of l ines o f the c a r b o n i. Ej *i, e n r i c h m e n t

o f the pos i t ion j was d e t e r m i n e d b y Eqn. 2 u s ing c a r b o n i as the r e fe rence

G l u c o s e E~ 4 = 5 5 ~ E~ 'l -- 10 E~ 3 = 10

R i b o s e E~, 3 = E~', 3 = 12 E f 2 = 53 ~ E,~, 3 = 5 El, 3 = 4

G l y c i n e El+2 = 5 2 E f I 5 A l a n i n e E~ 2. 53 E~ i = 4 , 5 E .2 4 3 A s p a r t a t e Ei ~2 = 5 3 E~ 3 = 6 E +4 7,5 3 U r i d i n e E~ '4 -- 6 E~ 4 = g

G l u t a m a t e E + z - 3 = 55 E~ i = 22 E~ 2 ~- 9 E g 2 ~- 9

E~ 3 2 = 5 5

E +~ = 5 3 a

E al = 17 5

" It was s u p p o s e d t ha t the e n r i c h m e n t o f the s econd i n t e r a c t i n g c a r b o n was negl igible .

suggests that besides the molecules labelled at the carbons 1 and 2, the molecules labelled only at the carbons 1 were synthesized in the cells, the conclu- sion should be drawn that the 'absolute' enrich- ment of the carbons 1 coincides with E~ 2 3 and in accordance with Scheme II is close to that of exogenous 13CO 2. Higher than in the case of carbons 3 and 4, enrichment of the carbons 2 and 5 indicates that part of the molecules of glutamate were synthesized through reductive carboxylic acid cycle. Simple calculations demonstrate that the best fit to the enrichment pattern of the glutamate (E~ ' t = 17% and E~ - I = 22%) is obtained assuming that about 30% of the molecules were synthesized through the reactions of the reductive carboxylic acid cycle, 70% being derived from oxaloacetate and exogenous acetate through the reactions of the Krebs cycle.

The value E~ - 4 - 3 = 53% indicates that in those molecules of glutamate where the carbons 4 were labelled, the enrichment of the carbons 5 was close to that of exogenous 13CO2. Taking into account also the practical coincidence of the enrichment of

, 2 ~ , 2 _,~.~ carbons 3 and 4 (E 3 - E 4 ~ 9%), the conclusion should be drawn that the carbons 4 and 5 (and 2, 3) in the glutamate molecules, synthesized through the reactions of the Krebs cycle, are practically unlabelled, derived directly from exogenous acetate.

Tyrosine In the 13C-NMR spectrum of the fraction which

contains tyrosine and glutamate, two groups of

tyrosine lines were clearly seen, one with the sing- let peak at 154 ppm and another with the singlet peak at 115 ppm. These lines correspond to the tyrosine atoms 6 + 8 and 7, respectively. Calcu-

-+ 7 lated enrichment of these positions, E6+ 8 = 54% and E~ -(6+8) = 50%, should by the logic of move- ment of carbons in the tyrosine-synthesizing path- way coincide with those of the positions 1 and 2 of erythrose 4-phosphate, and the values obtained indicate that erythrose 4-phosphate is synthesized through the transketolase reaction from fructose 6-phosphate.

All four nucleosides were separated from the fraction of RNA. Since the biosynthetic pathways of the purines (and pyrimidines) coincide (sup- ported also by the data obtained by us), only the t3C-NMR spectra of uridine and guanosine will be discussed.

Uridine The t3C-NMR spectrum of the uridine (Fig. 4)

shows that carbons 2, 3' and 4 have much higher levels of enrichment than the other positions in the molecule. The level of enrichment of carbon 4 was determined from the group of lines of carbon 5: E + 5 = 53% and that of carbon 5 was determined from the group of lines of carbon 4: E~ 4 = 7%. The enrichment of the positions 2, 5 and 6 was determined by Eqn. 2 using carbon 4 as a refer- ence. As the intensities of all the carbon lines in the natural-abundance 13C-NMR spectrum of uridine are practically equal, coefficients K4z, K45 and K46 in Eqn. 2 should be taken equal to 1. The

' C ' 2

4° ,'o C H E M I C A L

i

U R I D I N E C3'

C(~xNH 16 I

s' C.~. 3 .o ,c~ N, % • j / 0 ~ l

~i,, ~'C ,'N3' 2'Z' • ° gVg."

40 ,'oo ,% ,'o $ H I F T (p pa~]

Fig. 4. Proton-decoupled IsC-NMR spectrum of the labelled uridine from C. thiosulfatophilum.

317

levels of enrichment calculated were E~ '4 = 67, E5 .4 = 8 a n d JEff 4 = 6. The levels of enrichment of the carbons 4, 5 and 6 were practically equal to those of the positions 4, 3 and 2 of the labelled aspartate (see Table I), which is in good agreement with the uridine synthesis pathway from oxaloacetate and carbamyl phosphate.

The levels of enrichment of the carbons 5' (4%) and 4' (5%) in the ribose unit of the uridine coincide with those of the carbons 2 and 3 of the trioses, intermediates of the gluconeogenesis path- way, which is in agreement with the synthesis of the ribose 5-phosphate through the reactions of

polyglueose • • 1"

tyr°sine'*" ~dS~:CHOHCHOH -CliO ' t u ~ ~ ~ - - fructose 611~-P2

, , , y / / / \ POCH 2 CHOH-CHOH -CHOH(~ / / _ \

~ / * ~, kl_ I ~ glyeero 3-/' ,-* dihydroxy acetone P

dbulose 5-P HOOC-CttOH-Ctt~ OP ~glycine ,

alanine 2-phosphoglycerate

• o ~ * II ,

C H s C O O H ~ A c . - C o A r C H s C - C O O H .--~" phosphoenolpyruvate

citrate

isocitrate

"-+ ~o2 O II ,

HOOC-CH2CH2 -C-COOH ~

glutamate

HOOC-CH2 C-COOH --~aspartate il

malate

fumarate l

sucdnate CO2

succinyl-CoA

Scheme II. Main pathways and their directions, functioning according to the analysis of the labelling patterns of the separated metabolites in the cells of C thiosulfatophilum during heterotrophic growth. Organic-bound phosphates are abbreviated to -P.

318

the Calvin cycle (see Table I and Scheme II). This conclusion is supported also by the strong label- ling of the carbon 3' (53%), in accordance with carbon 3' being derived from the carbon 1 of the triosephosphates. But if the complete Calvin cycle functioned, i.e., if ribulose 1.5-diphosphate carboxylase reaction took place in the cells of C. thiosulfatophilum, levels of enrichment of the carbons 1' and 2' comparable to that of the carbon 3' should be expected. Low levels of enrichment of the carbons 1' and 2' can be explained by the assumption that the main bulk (85%) of the labelled ribose was synthesized through the ribose-phos- phate isomerase reaction in the reverse direction as in comparison with that expected in the case of functioning of the Calvin cycle (see Scheme II).

Guanosine The labelling pattern of the ribose unit of the

guanosine was similar to that of the labelled uridine. In the labelled guanine the positions 6 and 4 were enriched to about 50% and enrichment of the other positions was less than 10%. This is in agreement with guanine biosynthesis pathway in which the carbons 6 and 4 of guanine are derived from 13CO2 and the carboxyl group of glycine, respectively.

The polysaccharide fraction, separated from the cells consisted exclusively of polyglucose. The la- belling pattern of the glucose obtained has been extensively analysed elsewhere [15]; here we should like to point out only the results relevant to the subject of this paper. First of all, as shown in Table I, the enrichment of the carbons 3 and 4 in the glucose was 55%; the enrichment of the other carbons did not exceed 10%. These facts are in good agreement with the conclusion that the main bulk of the labelled glucose was synthesized through the gluconeogenesis pathway from the exogenous acetate and 13CO2.

All the data obtained allow us to propose the scheme of the main metabolic pathways and their directions functioning in C. thiosulfatophilum un- der the conditions of heterotrophic growth (Scheme II). It must be also pointed out that the presence of all the key enzymes needed for the functioning of the scheme have been demonstrated by Sirev~g [16], Smillie at al. [17], Evans et al. [8] and Beuscher and Gottschalk [ 18].

D i s c u s s i o n

The labelling patterns of the substances investigated, taking into account only the strongly labelled carbons, enrichment greater than 10%, are in good agreement with Scheme II. Thus our data - the enrichment patterns of the aspartate, gluta- mate and uridine show that ferredoxin-depen- dent CO2-fixation reactions of the postulated reductive carboxylic acid cycle do take place in the cells of C. thiosulfatophilum.

However, enrichment patterns of the studied metabolites indicate that reactions, additional to those shown in Scheme II, take place in the cells of C. thiosulfatophilum, giving rise to the minor enrichments (about 5-10%) of the substances (see Table I). Analysis demonstrated that the function- ing of either of the main CO2-fixation cycles - the Calvin cycle or the reductive carboxylic acid cycle - could lead to most of the minor enrichment of the carbons noted.

However, remarkably lower levels of enrich- ment of the carbons 1' and 2' in comparison with the carbons 3' in the ribose indicate that the direction of the ribose-phosphate isomerase reac- tion under the experimental conditions used is opposite to that expected when the Calvin cycle functions. If the Calvin cycle functioned in the cells, then it could be expected also that E2 +~ in glucose would be notably greater than E~ 3 in alanine, indicating the inflow of the products of the Calvin cycle into the gluconeogenesis pathway. But this was not observed. The facts mentioned should be considered as strong evidence against the functioning of the Calvin cycle in C. thiosulfa- tophilum during heterotrophic growth. Therefore the conclusion should be drawn that besides the reactions shown in Scheme II, the reductive carboxylic acid cycle rather than the Calvin cycle functions in the bacteria under the experimental conditions employed. Functioning of the reductive carboxylic acid cycle in the bacteria is in good agreement with the recently published results [19] demonstrating the presence of ATP-citrate lyase in the bacteria. Accepting the functioning of the re- ductive carboxylic acid cycle in the bacteria we should reconcile it with the postulated flows (see Scheme II) of substances meeting at the glutamate. Reconciliation could be reached by assuming that

quite elaborate time or spatial compartmentation of the reactions (directions) exist in the cells of C. thiosulfatophilum.

It should be also stated explicitly that func- tioning of the Calvin cycle during the autotrophic growth of the bacteria cannot be excluded by our data. Indeed, it has been assumed that high con- centrations of acetate inhibit the functioning of the Calvin cycle. If this is the reason for the non-functioning of the Calvin cycle during the heterotrophic growth of the bacteria, the absence of acetate in the culture medium during auto- trophic growth should lift the inhibition.

Enrichment of the carbons 2 determined by Eqn.l from the group of lines of the carbons 3 in the alanine (28%) (see Results and also Ref. 15) indicates that, in addition to the reactions of the reductive carboxylic acid cycle, other reactions of pyruvate synthesis should take place in the bacteria. In these reactions alanine molecules, en- riched only in the carbons 3 (additional to carbons 1) are synthesized. It was assumed in Ref. 15, following Ref. 20 that the reactions of the Entner- Doudoroff pathway were responsible for the synthesis of those glucose molecules labelled, ex- cept for carbons 3 and 4, only in the first and sixth positions. The same assumption explains the synthesis of the alanine molecules with the peculiarities of the labelling pattern noted above.

Analysis of the reaction mechanisms of the enzymes of the reductive carboxylic acid cycle showed that the enrichment pattern of the metabolites produced by this cycle depends on the stereospecificity of citrate lyase. If the a-keto- glutarate were synthesized from exogenous acetate and 13CO2 through the reactions of the reductive carboxylic acid cycle, and therefore strongly labelled at carbons 1, 2 and 5, the labelling pat- terns of the alanine and aspartate, products of the reductive carboxylic acid cycle, would depend on the type of the citrate lyase functioning in C. thiosulfatophilum. The analysis carried out showed that the labelling patterns of the alanine (higher level of enrichment in carbon 2 than in carbon 3)

319

and of the aspartate (higher enrichment in carbon 3 than in carbon 2) are in agreement with the functioning of the si-citrate lyase in the bacteria and incompatible with the functioning of the re- citrate lyase.

The data reported in the paper are in good agreement with recent results obtained by Fuchs et al. [21,22] using l aC-labelled compounds.

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