FEMS Microbiology Letters 241 (2004) 21–26
www.fems-microbiology.org
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
*
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany
Received 3 September 2004; received in revised form 21 September 2004; accepted 24 September 2004
First published online 8 October 2004
Edited by Dieter Jahn
Abstract
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 acetylCoA synthetase (ACD) were upregulated during periods of glucose consumption and acetate formation, whereas both AMPforming 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.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: 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 cat*
Corresponding author. Tel.: +49 431 880 4328; fax: +49 431 880
2194.
E-mail address: [email protected] (P. Schönheit).
alyzed 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].
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.09.033
22
C. Bräsen, P. Schönheit / FEMS Microbiology Letters 241 (2004) 21–26
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 acetylCoA. 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 (DOD578). DOD578 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 lmol substrate consumed or product formed per minute.
Acetyl-CoA synthetase (ADP-forming) (ACD)
(E.C. 6.2.1.13) was measured as described in [10].
Acetyl-CoA synthetase (AMP-forming) (ACS)
(E.C. 6.2.1.1.) was monitored as PPi and AMP dependent HSCoA release from acetyl-CoA according to Srere
et al. [11] with EllmanÕs thiol reagent, 5 0 5-dithiobis (2nitrobenzoic acid) (DTNB), by measuring the formation
of thiophenolate anion at 412 nm (e412 = 13.6
mM1 cm1). 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. 4.1.3.2.) 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. 1.1.1.47) was measured according to Johnsen et al. [1].
Acetate kinase (E.C. 2.7.2.1.) was measured as described in [2].
Phosphotransacetylase (E.C. 2.3.1.8.) 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.
C. Bräsen, P. Schönheit / FEMS Microbiology Letters 241 (2004) 21–26
1.8
6
Growth (∆OD578)
5
15
4
3
10
2
5
[Glucose, Acetate] (mM)
20
1
0
12
45
9
30
6
15
3
0
0
0
35
70
105
-1
60
ACS, MS (mU mg )
-1
ACD, GDH (mU mg )
0
Growth (∆OD578)
0.9
20
0.6
10
0
75
9
50
6
25
3
0
0
-1
0.0
ACS, MS (mU mg )
Glucose-adapted cells grew on acetate containing
medium initially (about 30 h) with a doubling time of
10 h up to an optical density (DOD578) 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
DOD578 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,
30
1.2
0.3
-1
3.2. Growth of glucose-adapted cells on acetate
40
1.5
ACD, GDH (mU mg )
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).
[Glucose, Acetate] (mM)
3.1. Growth of acetate-adapted cells on glucose
23
0
35
70
105
140
Time (h)
Fig. 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.
whereas increase of ACS activity was parallel to acetate
consumption (Fig. 2).
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 DOD578 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 DOD578 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).
140
Time (h)
Fig. 1. Growth of H. marismortui on glucose. Acetate-adapted cells
were used as inoculum. DOD578 (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).
3.4. 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 DOD578 of 2.5. Acetate
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C. Bräsen, P. Schönheit / FEMS Microbiology Letters 241 (2004) 21–26
5
30
3
20
2
10
1
0
60
15
40
10
20
5
0
0
0
30
60
90
-1
-1
ACD, GDH (mU mg )
0
ACS, MS (mU mg )
Growth (∆OD578)
4
[Glucose, Acetate] (mM)
40
120
Time (h)
Fig. 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.
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 for-
mation 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 [13–15]. 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
C. Bräsen, P. Schönheit / FEMS Microbiology Letters 241 (2004) 21–26
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. 1–3). 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 acetatespecific 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
25
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.
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