Abstract

During phototrophic growth on acetate and CO2Rhodospirillum rubrum 2R contained malate synthase but lacked isocitrate lyase. Acetate assimilation by R. rubrum cells was stimulated by pyruvate, propionate glyoxylate, CO2 and H2. Acetate photoassimilation by R. rubrum cells in the presence of bicarbonate was accompanied by glyoxylate secretion, which increased after addition of fluoroacetate and decreased after addition of malonate. When acetyl-CoA was incubated with pyruvate in cell-free extracts, citramalate was formed. Citramalate was also formed from propionyl-CoA and glyoxylate. The existence in R. rubrum of a CO2-dependent cyclic pathway of acetate oxidation to glyoxylate with citramalate as an intermediate is proposed. Inhibitor analysis of acetate and bicarbonate assimilation indicated that pyruvate synthase is not involved in acetate assimilation in R. rubrum. The possible anaplerotic sequences employed by R. rubrum during phototrophic growth on acetate are discussed.

Introduction

Many photosynthetic bacteria of the Rhodospirillaceae family can grow on acetate as the sole source of organic carbon [1]. Acetate catabolism is known to proceed via the tricarboxylic acid (TCA) cycle. However, growth on acetate as the sole source of carbon is impossible if C4 acids drained from the TCA cycle for biosynthetic reactions are not replenished. In many Rhodospirillaceae the replenishment of C4 acids is accomplished via the glyoxylate cycle with isocitrate lyase as a key enzyme. A large group of Rhodospirillaceae including Rhodospirillum rubrum and Rhodobacter sphaeroides lack isocitrate lyase but contain malate synthase [2]. Since these microorganisms do not possess the glyoxylate cycle, they must have other means to support net synthesis of TCA cycle intermediates from acetate. When R. rubrum grows anaerobically in the light on acetate, bicarbonate is necessary for growth from small inocula [3]. It was suggested that under these conditions CO2 fixation via the Calvin cycle may be important for the disposal of excess reducing power [4]. Carbon dioxide is also required under heterotrophic growth conditions for assimilation of several reduced organic substrates. Assimilation of propionate, for example, is connected with a carboxylation to succinate [5, 6]. Citramalate was detected as a major early product of [2-14C]acetate fixation by whole cells and cell-free extract of R. rubrum[7–9]. According to the data [10] citramalate is formed in R. rubrum via the following sequence of reactions: glyoxylate+propionyl-CoA→erythro-β-methylmalyl-CoA→mesaconyl-CoA→mesaconate→citramalate. Thus, although there is some evidence that R. rubrum can use anaplerotic sequences other than the glyoxylate cycle, the pathway of acetate metabolism has not been completely resolved. The aim of this research was to investigate the metabolic pathway of acetate assimilation in R. rubrum.

Materials and methods

Strains and culture conditions

Rhodospirillum rubrum 2R was obtained from the Culture Collection of the Department of Microbiology, Moscow State University. Cells were grown photoheterotrophically at 2000 lux and 28°C using the medium of Ormerod [11] in which acetate (1 g l−1) and NaHCO3 (3 g l−1) were used instead of malate. When the ability of the bacteria to grow on the medium with acetate as the only carbon source was studied, 250 ml screw-cap bottles, which contained approximately 25 ml of the medium, were used, and the gas phase was replaced by argon. CO2 evolved during the growth was removed using a KOH trap. Cultures from the early exponential growth phase (∼60 mg protein l−1) were harvested, washed twice, and suspended (0.3–1 mg protein ml−1) in the basal mineral medium lacking NaHCO3.

Preparation of cell-free extracts

For the preparation of cell-free extracts cells were harvested, washed with 50 mM Tris-HCl (pH 7.5), and disrupted by sonication (22 kHz, 3 min, 4°C). Debris was removed by centrifugation (40 000×g, 30 min, 4°C).

Whole cell studies

Assays of keto acid excretion and 14C substrate fixation by the cells of R. rubrum were performed in medical syringes at a light intensity of 3000 lux. Reaction medium contained acetate (5 mM), bicarbonate (5 mM) and inhibitors as indicated in Tables 2–4. Organic acids excreted during 6 h incubation of the cells were detected as 2,4-dinitrophenylhydrazones [12] after removing cells by centrifugation. The reaction of 14C substrate fixation was initiated by addition of NaH14CO3 (0.04 MBq) or [1-14C]acetate (0.02 MBq) to the cell suspension and stopped at fixed time intervals by filtration of 1 ml of cell suspension through 0.45 μm nitrocellulose filter.

2

Bicarbonate and acetate photoassimilation by R. rubrum cells

Substrates in the medium Fixation rate (nmol min−1 (mg protein)−1
*CO2+H2 6.3 
*CO2+Acetate 3.1 
*CO2+Acetate+H2 2.6 
*Acetate 15.3 
*Acetate+CO2 27.9 
*Acetate+CO2+H2 50.7 
*Acetate+propionate 15.6 
*Acetate+glyoxylate 42.7 
*Acetate+pyruvate 54.0 
*Acetate+CO2+citramalate 1.8 
*Acetate+CO2+mesaconate 6.8 
*Acetate+CO2+malonate 3.8 
*Acetate+CO2+fluoroacetate 3.4 
Substrates in the medium Fixation rate (nmol min−1 (mg protein)−1
*CO2+H2 6.3 
*CO2+Acetate 3.1 
*CO2+Acetate+H2 2.6 
*Acetate 15.3 
*Acetate+CO2 27.9 
*Acetate+CO2+H2 50.7 
*Acetate+propionate 15.6 
*Acetate+glyoxylate 42.7 
*Acetate+pyruvate 54.0 
*Acetate+CO2+citramalate 1.8 
*Acetate+CO2+mesaconate 6.8 
*Acetate+CO2+malonate 3.8 
*Acetate+CO2+fluoroacetate 3.4 

The concentration of all organic substrates was 5 mM, * indicates radioactively labeled substrates.

3

Glyoxylate secretion by R. rubrum cells under anaerobic conditions in the light

Medium contained Glyoxylate excretion (mmol h−1 (mg protein)−1
Acetate 0.051 
Acetate+CO2 0.084 
Acetate+fluoroacetate 0.425 
Acetate+malonate 0.032 
Medium contained Glyoxylate excretion (mmol h−1 (mg protein)−1
Acetate 0.051 
Acetate+CO2 0.084 
Acetate+fluoroacetate 0.425 
Acetate+malonate 0.032 

The concentrations of fluoroacetate and malonate were 1 and 5 mM respectively.

4

The effect of KCN on bicarbonate and acetate assimilation by R. rubrum cells

Substrates in the medium Fixation rate (nmol min−1 (mg protein)−1
*CO2+H2 6.2 
*CO2+H2+acetate 2.5 
*CO2+H2+KCN 0.9 
*CO2+H2+KCN+acetate 0.8 
*Acetate 15.3 
*Acetate+CO2+H2 38.0 
*Acetate+KCN 13.0 
*Acetate+CO2+H2+KCN 14.8 
*Acetate+pyruvate 33.0 
*Acetate+pyruvate+KCN 32.0 
*Acetate+propionate 16.3 
*Acetate+propionate+KCN 14.2 
Substrates in the medium Fixation rate (nmol min−1 (mg protein)−1
*CO2+H2 6.2 
*CO2+H2+acetate 2.5 
*CO2+H2+KCN 0.9 
*CO2+H2+KCN+acetate 0.8 
*Acetate 15.3 
*Acetate+CO2+H2 38.0 
*Acetate+KCN 13.0 
*Acetate+CO2+H2+KCN 14.8 
*Acetate+pyruvate 33.0 
*Acetate+pyruvate+KCN 32.0 
*Acetate+propionate 16.3 
*Acetate+propionate+KCN 14.2 

The concentrations of KCN, pyruvate, and propionate were 1, 5 and 5 mM, respectively; * indicates radioactively labeled substrates.

Enzyme assays

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31), PEP carboxykinase (EC 4.1.1.32), and PEP carboxytransphosphorylase (EC 4.1.1.38) were assayed according to [13], propionyl-CoA carboxylase (EC 6.4.1.3) according to [5], pyruvate kinase (EC 2.7.1.40) according to [14], NADP-malic enzyme (EC 1.1.1.40) according to [15], acetyl-CoA synthetase (EC 6.2.1.1) according to [16], succinate dehydrogenase (EC 1.3.99.1) according to [17], fumarate hydratase (EC 4.2.1.2) according to [18], malate dehydrogenase (EC 1.1.1.37) according to [19], isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) according to [20]. Citramalate lyase (EC 4.1.3.22) was assayed by the method of Buckel and Bobi [21] modified by addition of 0.5 mM CoA.

Analytical methods

Organic acids were separated by thin-layer chromatography and were developed in two solvent systems: n-butanol:5 N formic acid:water (10:3:10), and n-amyl alcohol:5 N formic acid (1:1). Keto acids were separated by thin-layer chromatography as 2,4-dinitrophenylhydrazones with n-butanol, ethanol, 0.5 N NH4OH (7:1:2) as solvent. Both acid types were identified by cochromatography with authentic organic compounds. Protein was measured according to Lowry [22].

Results and discussion

R. rubrum cells grown under phototrophic conditions on the medium with acetate and CO2 contained all enzymes of the TCA cycle. Malate synthase was the only detected enzyme of the glyoxylate shunt in R. rubrum. Isocitrate lyase could not be detected in cell-free extracts. Citramalate lyase, pyruvate kinase, PEP and propionyl-CoA carboxylases were also detected (Table 1).

1

Enzyme activities in R. rubrum cell-free extracts

Enzyme Specific activity (nmol min−1 (mg protein)−1
Acetyl-CoA synthetase 124.0 
Citrate synthase 16.6 
Aconitase 55.8 
Isocitrate dehydrogenase 62.6 
2-Oxoglutarate dehydrogenase 2.9 
Succinate dehydrogenase 24.0 
Fumarase 35.2 
Malate dehydrogenase 533.0 
Malic enzyme 3.8 
Malate synthase 57.9 
Isocitrate lyase <0.1 
Pyruvate kinase 3.9 
Citramalate lyase 8.4 
PEP carboxylase 4.7 
PEP carboxykinase 3.9 
PEP carboxytransphosphorylase <0.1 
Propionyl-CoA carboxylase 6.2 
Enzyme Specific activity (nmol min−1 (mg protein)−1
Acetyl-CoA synthetase 124.0 
Citrate synthase 16.6 
Aconitase 55.8 
Isocitrate dehydrogenase 62.6 
2-Oxoglutarate dehydrogenase 2.9 
Succinate dehydrogenase 24.0 
Fumarase 35.2 
Malate dehydrogenase 533.0 
Malic enzyme 3.8 
Malate synthase 57.9 
Isocitrate lyase <0.1 
Pyruvate kinase 3.9 
Citramalate lyase 8.4 
PEP carboxylase 4.7 
PEP carboxykinase 3.9 
PEP carboxytransphosphorylase <0.1 
Propionyl-CoA carboxylase 6.2 

Cells were grown with acetate and bicarbonate in the light.

On the basis of enzyme activity the operation in R. rubrum of an anaplerotic acetate assimilation cycle with citramalate as an intermediate is proposed. The pathway is shown in Fig. 1 and is designated the citramalate cycle (CM cycle). The initial steps of the cycle comprise conversion of acetate and pyruvate to glyoxylate and propionate with citramalate as an intermediate. The enzymes required for subsequent steps of the cycle were found in R. rubrum: propionyl-CoA carboxylase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase, PEP carboxylase which catalyzes decarboxylation of oxaloacetate to PEP and pyruvate kinase forming pyruvate (Table 1). Thereby, the first acceptor of acetyl-CoA (pyruvate) is regenerated, and the cycle is completed. The overall stoichiometry of the cyclic pathway is as follows: acetate→glyoxylate+4[H]. Thus, the proposed CM cycle would function in net oxidation of acetate to glyoxylate. Malate synthase catalyzes condensation of glyoxylate and an exogenous molecule of acetate to form malate. The latter would enter the TCA cycle and supply biosynthetic requirements of cells in C4 substrates. Therefore, the CM cycle is analogous to the glyoxylate cycle.

1

Proposed pathway of acetate oxidation to glyoxylate by R. rubrum as suggested by our results.

1

Proposed pathway of acetate oxidation to glyoxylate by R. rubrum as suggested by our results.

Our data indicate citramalate involvement in R. rubrum acetate metabolism. Citramalate could be actually synthesized via condensation of either acetate and pyruvate, or glyoxylate and propionate (Fig. 1). The rates of pyruvate and glyoxylate consumption in R. rubrum cell-free extracts in the presence of acetyl-CoA or propionyl-CoA were 2.0 and 4.8 nmol min−1 (mg protein)−1 respectively. The products of both processes were evaluated using thin-layer chromatography and a compound with the same Rf as authentic citramalate was identified using different solvent systems (data not shown). The inhibitory effect of citramalate or mesaconate on acetate assimilation (Table 2) also indicates the possibility of their involvement in this process. The shown decrease in the rate of acetate assimilation may occur due to the substitution of acetate with citramalate or mesaconate as intermediates of the CM cycle.

The following data provide additional evidence for CM cycle functioning in R. rubrum. Acetate photoassimilation by R. rubrum cell suspensions in the presence of fluoroacetate was accompanied by glyoxylate secretion (Table 3). Glyoxylate accumulation was increased after addition of bicarbonate. Addition of fluoroacetate caused inhibition of acetate assimilation (Table 2) and stimulation of glyoxylate secretion (Table 3). These data support the idea that glyoxylate is an end product of the proposed CM pathway and that the TCA cycle is a major mechanism of its utilization. Malonate considerably repressed glyoxylate secretion and acetate assimilation (Tables 2 and 3). This finding suggests an important role of succinate dehydrogenase, which is competitively inhibited by malonate in both the TCA and CM cycles.

R. rubrum could grow in acetate medium only if supplemented with bicarbonate, but not dimethylsulfoxide (0.1%) as an alternative electron acceptor (data not shown). It means that CO2 is required not for disposal of reducing equivalents. The rate of propionyl-CoA carboxylation in the medium with acetate alone is limited by a low concentration of endogenous CO2. It correlates with the low propionyl-CoA carboxylase affinity (Km= 5.3×10−3 M) for bicarbonate [5]. Thus, a high bicarbonate concentration in the medium is required for maintenance of growth and a high rate of acetate assimilation.

The simultaneous presence of H2 and CO2 increased the rate of acetate photoassimilation (Tables 2 and 4). Addition of cyanide, an inhibitor of ribulose-1,5-bisphosphate carboxylase [23], eliminated the stimulatory effect of CO2 and H2 (Table 4). However, the rate of acetate photoassimilation was restored after addition of pyruvate but not propionate (Table 4). It seems that the rate-limiting step of the cycle is in the proposed sequence of reaction between propionate and pyruvate. Pyruvate synthesized in the Calvin cycle may be incorporated into the CM cycle and thus would accelerate acetate assimilation (Fig. 1). This assumption is supported by the data showing a stimulatory effect of exogenous pyruvate on acetate assimilation in the absence of CO2, and by the absence of such an effect of propionate (Table 2). However, it should be pointed out that actual contribution of the Calvin cycle to acetate metabolism in R. rubrum is negligible. The observed rate of CO2 fixation under photomixotrophic and photoheterotrophic conditions is about 5–10% of that observed for acetate (Tables 2 and 4). It seems that the main pathways of acetate assimilation are CM and TCA cycles connected via malate synthase reaction (Fig. 2).

2

Metabolism of acetate in R. rubrum under light anaerobic conditions.

2

Metabolism of acetate in R. rubrum under light anaerobic conditions.

CO2 fixation by R. rubrum cell suspensions in the presence of H2 was inhibited by KCN (Table 4) and was not restored after addition of acetate. This indicates that, despite the presence of pyruvate in R. rubrum cells [24], this enzyme does not participate in acetate assimilation. This conclusion is confirmed by data showing that in the purple sulfur bacterium Thiocapsa roseopersicina, in which the existence of pyruvate synthase is well documented [6], bicarbonate fixation in the presence of inhibitors is completely restored after acetate addition. The relatively low ratio (0.05) of the CO2/acetate photoassimilation in the medium containing acetate, bicarbonate and H2 (Table 2) is additional evidence of little or no participation of reductive carboxylation of acetate in acetate assimilation in R. rubrum. The corresponding ratio for T. roseopersicina is 1/1 or higher [6].

All the data of this study are consistent with the operation of the CM cycle as an anaplerotic sequence of reactions for acetate assimilation in photoheterotrophically grown R. rubrum.

Acknowledgements

This work was supported by the Russian Foundation for Basic Research.

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