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Regulation of acetate and acetyl-CoA converting enzymes during growth on acetate and/or glucose in the halophilic archaeon Haloarcula marismortui

Christopher Bräsen, Peter Schönheit
DOI: http://dx.doi.org/10.1016/j.femsle.2004.09.033 21-26 First published online: 1 December 2004


Haloarcula marismortui formed acetate during aerobic growth on glucose and utilized acetate as growth substrate. On glucose/acetate mixtures diauxic growth was observed with glucose as the preferred substrate. Regulation of enzyme activities, related to glucose and acetate metabolism was analyzed. It was found that both glucose dehydrogenase (GDH) and ADP-forming acetyl-CoA synthetase (ACD) were upregulated during periods of glucose consumption and acetate formation, whereas both AMP-forming acetyl-CoA synthetase (ACS) and malate synthase (MS) were downregulated. Conversely, upregulation of ACS and MS and downregulation of ACD and GDH were observed during periods of acetate consumption. MS was also upregulated during growth on peptides in the absence of acetate. From the data we conclude that a glucose-inducible ACD catalyzes acetate formation whereas acetate activation is catalyzed by an acetate-inducible ACS; both ACS and MS are apparently induced by acetate and repressed by glucose.

  • Haloarcula marismortui
  • Acetate formation
  • Acetate activation
  • ADP-forming acety-CoA synthetase
  • AMP-forming acetyl-CoA synthetase
  • Overflow metabolism
  • Catabolite repression

1 Introduction

Various halophilic archaea, including Haloarcula marismortui, grow on glucose, which is degraded via a modified, semiphosphorylated Entner–Doudoroff (ED) pathway [1]. It has been shown that during exponential growth on glucose significant amounts of acetate were formed [2]. Recent studies indicate that the formation of acetate from acetyl-CoA in halophilic archaea is catalyzed by an ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + Pi⇆ acetate + ATP + CoA). This unusual synthetase was found in all acetate-forming archaea, including anaerobic hyperthermophiles, and represents a novel mechanism in prokaryotes of acetate formation and ATP synthesis. In anaerobic hyperthermophilic archaea, e.g. Pyrococcus furiosus, ACD represents the major energy conserving reaction during sugar, pyruvate and peptide metabolism [3, 4]. In contrast to the archaeal one-enzyme mechanism, all bacteria use the “classical” two-enzyme mechanism for acetyl-CoA conversion to acetate, involving phosphate acetyltransferase (PTA) and acetate kinase (AK) [5].

Several haloarchaea, including H. marismortui, Haloferax volcanii and Halorubrum saccharovorum have been reported to grow on acetate as substrate. The metabolism of acetate is initiated by its activation to acetyl-CoA. Recently, we provided first evidence that acetate activation to acetyl-CoA in haloarchaea is catalyzed by an AMP-forming acetyl-CoA synthetase (ACS) (acetate + ATP + CoA → acetyl-CoA + AMP + PPi) [2]. ACS is the major enzyme of acetate activation for most acetate-utilizing organisms from all three domains of life [6]. Only few bacteria, e.g. Corynebacterium glutamicum, and also the acetoclastic methanogenic archaeon Methanosarcina ssp. activate acetate to acetyl-CoA via the AK/PTA couple [7, 8]. Thus, the AK/PTA pathway can operate reversibly in vivo, i.e. both in the direction of acetate formation and also in the direction of acetate activation. In contrast, first analyses suggest that in haloarchaea ACD, the archaeal counterpart to the AK/PTA couple, operates in vivo in the direction of acetate formation, although the enzyme catalyzes a reversible reaction in vitro.

To further elucidate the physiological role and to get first insights into the substrate dependent regulation of acetate and acetyl-CoA converting enzymes in haloarchaea we carried out substrate shift experiments with H. marismortui, and analyzed growth on glucose, acetate, glucose/acetate mixtures and peptides. During growth activity profiles of acetate and acetyl-CoA converting enzymes, ACD and ACS, as well as of glucose dehydrogenase, the first enzyme in glucose degradation via the modified ED pathway, were analyzed. In addition, activities of malate synthase, one key enzyme of glyoxylate cycle, proposed to be operative in haloarchaea [2, 9], were determined.

2 Materials and methods

2.1 Growth of H. marismortui on glucose, acetate, glucose/acetate mixtures and on peptides

Haloarcula marismortui was grown aerobically at 37 °C on a complex medium containing yeast extract, casaminoacids and additionally glucose and/or acetate as described previously [2]. For growth on glucose/acetate mixture this medium was supplemented with 12.5 mM glucose and 30 mM acetate. Growth on peptides was performed on the complex medium in the absence of acetate and glucose. Growth experiments were carried out in 2 l fermentors (fairmen tec, Germany) with a stirrer velocity of 500 rpm and a throughput of compressed air of 600 ml per minute. Growth was followed by measuring the optical density at 578 nm (ΔOD578). ΔOD578 of 1 corresponded to a protein content of 0.5–0.6 mg/ml. Glucose and acetate were determined enzymatically as described in [2].

2.2 Preparation of cell extracts

At various growth phases cells of H. marismortui (100–200 ml of the culture) were harvested and cell extracts were prepared as described in [2]. Protein was determined by the Bradford method using bovine serum albumin as a standard.

2.3 Determination of enzyme activities

All enzyme assays were performed under aerobic conditions at 37 °C in cuvettes filled with 1 ml assay mixture. The auxiliary enzymes were generally added shortly before start of the reaction and it was ensured that these enzymes were not rate limiting. One unit (1 U) of enzyme activity is defined as 1 μmol substrate consumed or product formed per minute.

  • Acetyl-CoA synthetase (ADP-forming) (ACD) (E.C. was measured as described in [10].

  • Acetyl-CoA synthetase (AMP-forming) (ACS) (E.C. was monitored as PPi and AMP dependent HSCoA release from acetyl-CoA according to Srere et al. [11] with Ellman's thiol reagent, 5′5-dithiobis (2-nitrobenzoic acid) (DTNB), by measuring the formation of thiophenolate anion at 412 nm (ε412= 13.6 mM−1 cm−1). The assay mixture contained 100 mM Tris–HCl, pH 7.5, 1.25 M KCl, 2.5 mM MgCl2, 0.1 mM DTNB, 1 mM acetyl-CoA, 2 mM AMP, 2 mM PPi and extract.

  • Malate synthase (E.C. was monitored in a modified assay according to Serrano et al. [12] with DTNB. The assay mixture contained 20 mM Tris–HCl, pH 8.0, 3 M KCl, 30 mM MgCl2, 0.1 mM DTNB, 0.2 mM acetyl-CoA, 0.5 mM glyoxylate and extract.

  • Glucose dehydrogenase (E.C. was measured according to Johnsen et al. [1].

  • Acetate kinase (E.C. was measured as described in [2].

  • Phosphotransacetylase (E.C. was monitored as Pi dependent HSCoA release from acetyl-CoA with DTNB [11]. The assay mixture contained 100 mM Tris–HCl, pH 7.5, 3 M KCl, 30 mM MgCl2, 0.1 mM DTNB, 1.5 mM acetyl-CoA, 5 mM KH2PO4 and extract.

3 Results

To investigate the physiological function and regulation of enzymes related to acetate and acetyl-CoA metabolism, cells of H. marismortui pregrown on different substrates were shifted to medium containing acetate and/or glucose and peptides, respectively, and activity profiles of ACD, ACS, GDH and MS were analyzed.

3.1 Growth of acetate-adapted cells on glucose

After a lag phase cells grew exponentially and glucose was completely consumed. Parallel to glucose consumption significant amounts of acetate were formed. In this period the activities of both GDH and ACD increased, whereas the activities of ACS and MS, which were active in acetate adapted cells, were completely downregulated. In the stationary phase, the excreted acetate was completely reconsumed and both ACS and MS activities increased, whereas GDH and ACD activities decreased (Fig. 1).

Figure 1

Growth of H. marismortui on glucose. Acetate-adapted cells were used as inoculum. ΔOD578 (filled squares), glucose concentration (filled triangles), acetate concentration (filled circles); enzyme activities: ACD (filled diamonds), GDH (inverse filled triangles), ACS (open circles), MS (open triangles).

3.2 Growth of glucose-adapted cells on acetate

Glucose-adapted cells grew on acetate containing medium initially (about 30 h) with a doubling time of 10 h up to an optical density (ΔOD578) of 1. In this growth phase no acetate consumption was observed and cells grew on peptides present in the medium. After this period, cells grew with a reduced growth rate up to ΔOD578 of 1.8 and acetate was completely consumed. During acetate consumption ACD and GDH activities decreased, whereas ACS and MS activities, which could not be detected in glucose-adapted cells, increased. Increase of MS activity started during growth on peptides, whereas increase of ACS activity was parallel to acetate consumption (Fig. 2).

Figure 2

Growth of H. marismortui on acetate. Glucose-adapted cells were used as inoculum. The same symbols were used as described in the legend of Fig. 1.

3.3 Growth on glucose/acetate mixtures

Cells of H. marismortui adapted to yeast extract and casaminoacids were transferred to medium containing both glucose and acetate. The cells showed diauxic growth with sequential utilization of first glucose and second acetate. In the first growth phase cells grew up to ΔOD578 of 4.0 and glucose was consumed. After glucose consumption and a short lag phase cells entered the second growth phase, in which acetate was metabolized and cells grew to a final ΔOD578 of 5.2. In the first growth phase parallel to glucose consumption ACD and GDH activities increased, whereas ACS activity could not be detected and MS activity was completely downregulated. In the second growth phase parallel to acetate utilization ACS and MS activities increased and ACD and GDH activities decreased (Fig. 3).

Figure 3

Growth of H. marismortui on glucose/acetate mixture. Cells adapted to complex constituents in the absence of glucose and acetate were used as inoculum. The same symbols were used as described in the legend of Fig. 1.

3.4 Glucose-adapted cells on peptides

Glucose-adapted cells were shifted to medium containing 0.25% yeast extract and 0.5% casaminoacids in the absence of both glucose and acetate. The cells grew with doubling time of 13 h up to ΔOD578 of 2.5. Acetate formation could not be detected. During exponential growth ACD (from 60 to 20 mU/mg) and GDH (from 80 to 40 mU/mg) decreased, and MS activity, which initially could not be detected, increased up to 20 mU/mg. ACS activity could not be detected during exponential growth phase, but increased during the stationary phase (13 mU/mg).

4 Discussion

In this paper we analyzed the physiological role of acetate and acetyl-CoA converting enzymes (ACD, ACS) in H. marismortui and give first evidence for substrate specific regulation of these enzymes as well as for GDH and MS. The data are discussed in comparison with known bacterial systems.

4.1 Acetate formation in H. marismortui is catalyzed by ACD as part of “overflow” metabolism

During growth on glucose and on glucose/acetate mixtures, both ACD and GDH activities increased parallel to phases of glucose consumption and acetate formation (Figs. 1 and 3). Conversely, both activities decreased during growth on acetate or peptides. These data and the absence of AK/PTA indicate that the formation of acetate in Haloarcula is catalyzed by ACD. The physiological role of acetate formation in H. marismortui and its regulation during aerobic growth on glucose is not understood; acetate formation might be part of an “overflow” metabolism, which has been studied in some detail in various bacteria, e.g. Escherichia coli and Bacillus subtilis. As in H. marismortui, both bacteria excrete acetate during aerobic growth on excess glucose and reutilize it in the stationary phase [1315]. It has been speculated that excretion of acetate occurs under conditions when the rate of glycolysis exceeds that of subsequent pathways, e.g., citric acid cycle and respiration required for complete oxidation of glucose [16]. Under these conditions acetyl-CoA is converted to acetate and excreted. In accordance with this view, transcriptional analyses with both E. coli and B. subtilis indicate glucose-specific induction of glycolytic genes and repression of genes of the citric acid cycle and of respiration [17, 18]. A similar glucose-specific transcriptional regulation, i.e. upregulation of glycolytic genes of the modified Entner–Doudoroff pathway, and downregulation of some genes of the citric acid cycle and of respiration has recently been reported for the halophilic archaeon H. volcanii[19]. Thus, in haloarchaea a glucose-specific overflow metabolism resulting in acetate formation is likely. Acetate formation in E. coli and B. subtilis involves the bacterial two-enzyme mechanism via PTA and AK, whereas in Haloarcula acetate formation is catalyzed by ACD, the archaeal one-enzyme mechanism. In both, E. coli and B. subtilis, glucose was found to induce the encoding pta and ack genes indicating coordinate regulation of glycolysis and acetate formation [13, 17, 20]. So far, transcriptional regulation of the acetate-forming ACD in the archaeon H. marismortui has not been analyzed. However, the coordinate regulation of GDH and of ACD activity suggests a similar glucose-specific transcriptional regulation of both glycolysis by the modified Entner–Doudoroff pathway and of acetate formation by ACD.

During aerobic growth on peptides H. marismortui did not form acetate and ACD activity was downregulated. In this respect Haloarcula differs from the bacterium E. coli, which forms significant amounts of acetate during aerobic growth on peptides in the course of an “overflow” metabolism [21]. H. marismortui also differs from the anaerobic hyperthermophilic archaeon P. furiosus and other anaerobic, hyperthermophilic archaea, which form high amounts of acetate by means of ACD during anaerobic growth on both sugars and peptides. During anaerobic peptide and sugar fermentation of P. furiosus, acetate formation by ACD represents the major site of ATP formation via substrate level phosphorylation [3]; in contrast, during aerobic degradation of sugars and peptides by H. marismortui most energy is conserved by electron transport phosphorylation in the respiratory chain and thus acetate formation by ACD is less important or dispensable. Thus, acetate formation by ACD in Haloarcula appears to be restricted to sugar metabolism in course of an “overflow” metabolism.

4.2 Acetate activation to acetyl-CoA in H. marismortui is catalyzed by ACS

This was concluded from the upregulation of ACS activity parallel to acetate consumption (Figs. 1, 2, 3). A role of ACD in acetate activation could be ruled out since the ACD was downregulated during periods of acetate consumption. Thus, ACD in Haloarcula is operating in vivo only in direction of acetate formation. ACS is also the most common mechanism of acetate activation in bacteria where it is strictly regulated; e.g. in E. coli and B. subtilis, the acs gene is induced by acetate and repressed by glucose [17, 22]. In Haloarcula, upregulation of ACS activity by acetate and downregulation by glucose suggest a similar regulation on the transcriptional level as reported for bacteria. It should be noted that in contrast to E. coli and B. subtilis, the bacterium C. glutamicum activates acetate by an acetate induced AK/PTA pathway [7].

Activity of MS, a key enzyme of the glyoxylate cycle, was upregulated during periods of acetate consumption in H. marismortui together with ACS suggesting acetate-specific coordinate regulation of ACS and the anaplerotic glyoxylate cycle. Both MS and ACS activity were down regulated by glucose. Coordinate acetate-specific induction of genes of the enzymes of acetate activation (see above) and of the glyoxylate pathway has been reported for several bacteria, including E. coli and C. glutamicum[7, 17]. Recently, first evidence for induction of both malate synthase and isocitrate lyase genes by acetate has been given for halophilic archaeon H. volcanii[9].

However, MS activity rather than ACS activity was also upregulated during exponential growth on peptides in the absence of acetate indicating that regulation of MS is more complex and not restricted to acetate. A role of MS (and of glyoxylate cycle) in peptide metabolism might be explained by the fact that many amino acids are degraded to acetyl-CoA, which would require a functional glyoxylate pathway for anabolism. The increase of ACS activity, observed in the stationary phase during growth on peptides, cannot be explained so far, it might be due to a general stress response of stationary phase cells [21, 23].

4.3 Glucose specific catabolite repression in H. marismortui

Haloarcula marismortui showed diauxic growth on glucose/acetate mixtures with glucose as preferred substrate indicating some sort of catabolic repression of acetate utilization by glucose. Glucose specific catabolite repression has not been analyzed in archaea so far. In bacteria the molecular basis of carbon catabolite repression by glucose has been studied in detail, e.g in E. coli and B. subtilis[24]. During growth of C. glutamicum on glucose/acetate mixtures monophasic growth was described with simultaneous consumption of acetate and glucose, whereas in Azotobacter vinelandii acetate is the preferred substrate. The regulatory principles behind these features are currently investigated [7, 25].

Further studies are necessary to substantiate the proposed substrate specific regulation of acetate forming and acetate activating enzymes, ACD and ACS, in relation to acetate and glucose metabolism on the transcriptional level. These studies, which require the purification and identification of the encoding genes of the ACD [10] and ACS from H. marismortui are in progress.


  • Editor: Dieter Jahn


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