Download Acetate formation in the photoheterotrophic bacterium Chloroflexus

RESEARCH LETTER
Acetate formation in the photoheterotrophic bacterium
Chloroflexus aurantiacus involves an archaeal type
ADP-forming acetyl-CoA synthetase isoenzyme I
€ nheit
Marcel Schmidt & Peter Scho
€r Allgemeine Mikrobiologie, Christian-Albrechts-Universit€
Institut fu
at Kiel, Kiel, Germany
€nheit, Institut
Correspondence: Peter Scho
€r Allgemeine Mikrobiologie, Christianfu
Albrechts-Universit€
at Kiel, Am Botanischen
Garten 1-9, D-24118 Kiel, Germany.
Tel.: +49 431 880 4328/4330;
fax: +49 431 880 2194;
e-mail: [email protected]
Received 20 September 2013; revised 21
October 2013; accepted 21 October 2013.
Final version published online 13 November
2013.
DOI: 10.1111/1574-6968.12312
MICROBIOLOGY LETTERS
Editor: Dieter Jahn
Keywords
Chloroflexus aurantiacus; acetate formation;
adenosine diphosphate-forming acetyl-CoA
synthetase; acetate kinase;
phosphotransacetylase; Bacteria.
Abstract
The bacterium Chloroflexus aurantiacus excreted significant amounts of acetate
during photohetero trophic growth on glucose and in resting cell suspensions.
Up to 1.5 mol acetate per mol glucose were formed. In acetate-forming cells,
the activities of phosphotransacetylase and acetate kinase, usually involved in
acetate formation in Bacteria, could not be detected; instead, the cells contained
an acetyl-CoA synthetase (ADP-forming) (ACD) (acetyl-CoA + ADP +
Pi ? acetate + ATP + CoA), an enzyme so far reported in prokaryotes to be
specific for acetate-forming Archaea. ACD, which was induced 10-fold during
growth on glucose, was purified and the encoding gene was identified as
Caur_3920. The recombinant enzyme, a homotetrameric 300-kDa protein composed of 75-kDa subunits, was characterized as functional ACD. Substrate specificities and kinetic constants for acetyl-CoA/acetate and other acyl-CoA esters/
acids were determined, showing similarity of the C. aurantiacus ACD to archaeal
ACD I isoenzymes, which are involved in acetate formation from sugars. This is
the first report of a functional ACD involved in acetate formation in the domain
of Bacteria.
Introduction
Acetate is an important product of fermentation processes
in many anaerobic and facultative prokaryotes of both the
bacterial and the archaeal domains. Analyses of the mechanism of acetate formation from acetyl-CoA indicate
differences in Bacteria and Archaea. All acetate-forming
Bacteria analyzed so far, catalyze acetate formation from
acetyl-CoA by the classical mechanism involving the two
enzymes phosphotransacetylase (PTA) and acetate kinase
(AK). In contrast, in all acetate-forming Archaea, including anaerobic hyperthermophiles and aerobic halophiles,
the conversion of acetyl-CoA to acetate is catalyzed by
one enzyme, an acetyl-CoA synthetase (ADP-forming)
(ACD; Sch€afer et al., 1993). ACD catalyzes the conversion
of acetyl-CoA to acetate and couples this reaction with
the synthesis of ATP from ADP and Pi via substrate level
phosphorylation
(acetyl-CoA + ADP + Pi ? acetate +
ATP + HSCoA). Thus, the mechanism of acetate formaFEMS Microbiol Lett 349 (2013) 171–179
tion from acetyl-CoA in prokaryotes appears to be
domain-specific (Sch€afer et al., 1993; Br€asen et al., 2008).
ACD is also present in eukaryotic protists, e.g. Entamoeba histolytica and Giardia lamblia (Reeves et al., 1977;
Sanchez & M€
uller, 1996), and ACD homologs have been
found in many bacterial genomes (Sanchez et al., 2000).
The metabolic functions of these putative ACDs have not
been studied so far. Several Bacteria, including the phototroph Chloroflexus aurantiacus and the syntrophs Pelotomaculum thermopropionicum and Syntrophus aciditrophicus,
have been reported to generate acetate in their metabolism,
but lack genes for AK and PTA. However, these Bacteria
contain one or more homologs of ACD genes (Kondratieva
et al., 1992; McInerney et al., 2007; Kosaka et al., 2008;
Tang et al., 2011), suggesting that these putative ACDs
might have a functional role in acetate formation.
In this work we studied the formation of acetate and
the enzyme involved in the photoheterotrophic bacterium
C. aurantiacus. It is shown that C. aurantiacus ferments
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Published by John Wiley & Sons Ltd. All rights reserved
€nheit
M. Schmidt & P. Scho
172
glucose to high amounts of acetate and that acetate formation is catalyzed by an ACD. The C. aurantiacus ACD
was purified and the encoding gene identified. The ACD
showed similar substrate specificity as ACD isoenzymes I
from Archaea. This is the first report of a functional ACD
involved in acetate formation in the domain of Bacteria.
It is proposed that C. aurantiacus acquired its ACD via
lateral gene transfer from Archaea.
Materials and methods
Growth conditions
Chloroflexus aurantiacus strain J-10-fl (DSM 635; Pierson
& Castenholz, 1974) was obtained from the Deutsche
Sammlung f€
ur Mikroorganismen und Zellkulturen
(DSMZ). Chloroflexus aurantiacus was grown under
anaerobic conditions at pH 8.3 and at 55 °C in 1-L bottles filled with 1000 mL medium with N2 as gas phase.
The bottles were shaken at 35 r.p.m. and illuminated with
two 40-W reflector lamps at 600–800 lux. Medium for
growth of C. aurantiacus was prepared according to
Herter et al. (2001) with a few modifications. The medium contained (per liter): 1 g yeast extract, 2.5 g casamino
acids, 3.33 g glycylglycine, 100 mL solution A, 0.5 mL
solution B, pH 8.3, 1.25 mL solution C, 1 mL solution D
and 20 mM glucose. Solution A contained (per liter): 1 g
EDTA, 0.6 g CaSO4H2O, 1 g MgSO47H2O, 0.08 g NaCl,
1.11 g Na2HPO4. Solution B contained (per liter):
2.28 g MnSO47H2O, 0.5 g ZnSO47H2O, 0.5 g H3BO3,
0.025 g CuSO45H2O, 0.025 g Na2MoO42H2O, 0.045 g
CoCl26H2O. Solution C contained (per liter): 0.08 g
cobalamin, 0.024 g thiamine hydrochloride, 0.4 g niacin,
0.04 g 4-aminobenzoic acid, 0.08 g biotin, 0.04 g calciumD-pantothenic acid, 0.008 g pyridoxal hydrochloride, 0.4 g
myoinosite, 0.008 g folic acid, and the pH was adjusted to
pH 7 with NaOH. Solution D consisted of a filter-sterilized solution of 2.9 g L 1 FeCl36H2O. The medium was
autoclaved and then filter-sterilized solutions C and D,
and glucose were added. Growth was monitored by
measuring the optical density at 600 nm (DOD600). An
DOD600 of 1 corresponded to a protein content of
0.72 mg mL 1 as determined by the Biuret method.
Acetate and glucose were determined enzymatically (Kunst
et al., 1986; Sch€afer et al., 1993).
Preparation of cell extracts
For determination of enzyme activity, cells of the stationary phase of growth were harvested by centrifugation
(1100 g, 8 °C, 20 min) and suspended in 100 mM
Hepes-KOH pH 7.5 and 5 mM MgCl2. Cells were disrupted by passing four times through a French pressure
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
cell at 14 000 psi. Cell debris was removed by centrifugation for 120 min at 4 °C with 100 000 g. Protein was
determined by the Bradford method.
Cell-suspension experiments
Cell suspensions of C. aurantiacus were prepared after
growth on media (see above) containing glucose but
omitting yeast extract. Cells of the exponential phase of
growth were harvested by centrifugation and washed
three times in the same volume of suspension buffer
(100 mM Hepes-KOH pH 8.5 with 10% Solution A).
Suspended cells were incubated at 55 °C for 45 min to
degrade potentially accumulated reserve substances,
washed once and then suspended in the same buffer at a
protein concentration of 13 mg mL 1. Cell-suspension
experiments were performed in 100-mL bottles filled with
a 20-mL cell suspension at 55 °C, under anaerobic conditions (N2 gas phase) in the dark.
Purification of ACD from C. aurantiacus
(Caur-ACD)
Cell extracts was prepared from 96 g wet weight cells
obtained after photoheterotrophic growth on glucose and
was incubated at 70 °C for 10 min and centrifuged for
45 min at 100 000 g and 4 °C. The supernatant was
adjusted to 0.5 M (NH4)2SO4 followed by an additional
centrifugation step, and was applied to a Phenyl Sepharose 26/10 HiLoad column equilibrated with 100 mM
Hepes-KOH pH 7.5 containing 5 mM MgCl2, 500 mM
(NH4)2SO4 and 10% (v/v) glycerol. Protein was desorbed
by linear gradient to 200 mM (NH4)2SO4. Fractions containing the highest ACD activity eluting at 280–230 mM
(NH4)2SO4 were pooled, diluted 50-fold in buffer A
(50 mM Hepes-KOH, pH 8.0, containing 5 mM MgCl2
and 10% glycerol) and applied to a Q-Sepharose 16/10column equilibrated with buffer A. The column was
washed with buffer A and protein was eluted by increasing NaCl concentrations up to 1 M. The highest ACD
activity eluted at 140–180 mM NaCl. The fractions with
the highest activity were concentrated by Amicon ultrafiltration (cutoff 30 kDa) to a final volume of 1 mL and
applied to a Superdex 200 HiLoad column (1.6 9 60 cm)
equilibrated with 100 mM Tris–HCl pH 7.5, 150 mM
NaCl, 5 mM MgCl2 and 10% glycerol (buffer B). Isocratic
elution was performed with 120 mL buffer B. Fractions
with the highest activity were diluted 50-fold in buffer A
and applied to Mono-Q-Sepharose column (1 mL) equilibrated with buffer A. The column was washed and protein was eluted with increasing NaCl concentrations up to
170 mM. The highest ACD activity eluted at 115 mM
NaCl. At this step, ACD was almost pure as analyzed by
FEMS Microbiol Lett 349 (2013) 171–179
173
ADP-forming acetyl-CoA synthetase in Chloroflexus
SDS PAGE. The gene encoding ACD was identified by
matrix-assisted laser desorption-time of flight (MALDITOF) mass spectrometry (Schaffer et al., 2001).
Cloning and expression of Caur-ACD and
purification of the recombinant enzyme
Caur_3920 was amplified from genomic DNA of C. aurantiacus by PCR and cloned into pET19b by two restricition sites (NdeI and XhoI) created with the primers: 5′
CCCATCATATGCTAGAAGC and 5′CTCGAGCATTAG
TTGAGGA (restriction sites are underlined). For expression of Caur_3920 the vector pET19bCaur_ACD was
transformed into Escherichia coli BL21(DE3) codon plus
RIL (Stratagene). Growth was performed in Luria–Bertani
medium with chloramphenicol (34 lg mL 1) and carbenicillin (100 lg mL 1) at 37 °C. Expression was induced
by the addition of 1 mM isopropyl-1-thio-b-D-galactopyranoside at an OD600 of 0.8. After 4 h of further growth,
the cells were harvested by centrifugation (20 min at 8 °C
and 4500 g) and resuspended in 100 mM Tris–HCl, pH
8.2, containing 300 mM NaCl and 10 mM imidazole
(buffer C). Cells were disrupted by sonication followed by
centrifugation at 100 000 g for 90 min. The supernatant
was applied to a Nickel-nitrilotriacetic acid (Ni-NTA)
column equilibrated with buffer C. Protein was eluted
with increasing concentrations of imidazole. ACD-containing fractions eluted at 250 mM imidazole were concentrated by ultrafiltration to a total volume of 1.2 mL
(30-kDa cutoff) and applied to a Superdex 200 HiLoad
16/60 column equilibrated with 100 mM Tris–HCl pH
7.5 and 150 mM NaCl. Protein was eluted by an isocratic
flow, yielding pure protein.
Enzyme assays
ACD activity was measured at 55 °C in both reaction
directions. In the direction of acetate formation, ACD
activity was followed as ADP- and Pi-dependent release of
HSCoA from acetyl-CoA with Ellman’s thiol reagent, 5,5dithiobis (2-nitrobenzoic acid) (DTNB), by measuring
the formation of thiophenolate anion at 412 nm (e412 =
13.6 mM 1 cm 1) (Srere et al., 1963). The assay mixture
(1 mL) contained 100 mM Hepes-KOH pH 7.5, 0.1 mM
DTNB, 5 mM MgCl2, 2 mM ADP, 0.1 mM acetyl-CoA,
20 mM KH2PO4. In the direction of acetate activation,
ACD activity was measured as HSCoA and ATP-dependent formation of ADP by coupling the reaction with the
oxidation of NADH via pyruvate kinase (PK) and lactate
dehydrogenase (LDH). The assay contained 100 mM
Hepes-KOH, 5 mM MgCl2, 2.5 mM PEP, 0.3 mM
NADH, 4 U PK, 6 U LDH, 10 mM acetate, 2 mM ATP,
1 mM HSCoA. The pH- and temperature-dependence of
FEMS Microbiol Lett 349 (2013) 171–179
recombinant enzyme was monitored with Ellman’s thiol
reagent. The temperature-dependence was determined
between 20 and 85 °C adjusting the pH of the buffer to
7.5 at the specific temperature. The pH-dependence was
determined between pH 5.0 and 8.5 using piperazine (pH
5.0–6.0), MES (pH 6.0–7.0), HEPES (pH 7.0–8.0) or glycylglycine (pH 8.0–8.5). Phosphotransacetylase activity
was measured as Pi-dependent release of HSCoA from
acetyl-CoA with DTNB using the same assay as described
above for ACD but omitting ADP. Activity of AK was
determined as described in Sch€afer et al. (1993).
Results
Acetate formation from glucose by
C. aurantiacus
Chloroflexus aurantiacus was grown photoheterotrophically on glucose (20 mM) in the presence of casamino acids
and yeast extract under a 100% N2 gas phase. The cells
grew with a doubling time of about 19 h to a cell density
DOD600 of 7.5. During growth, glucose (12 mM) was
consumed and, in parallel, acetate (about 8 mM) was
formed (Fig. 1a). In the absence of glucose, cells grew to
an DOD600 of 4 with a doubling time of 22 h and about
2.5 mM acetate was formed from complex constituents
(Fig. 1b). To prove acetate formation from glucose, resting cells of glucose-grown C. aurantiacus were incubated
in the dark in the absence of complex constituents. Under
these conditions the cells consumed glucose and formed
acetate at a ratio of 1.5 mol acetate per mol glucose
(Fig. 2), indicating that acetate is the main product of
C. aurantiacus fermentation of glucose.
Acetate formation in C. aurantiacus is
catalyzed by an ACD
Extracts of glucose-grown cells were analyzed for enzyme
activities of acetate formation from acetyl CoA. Activities
of PTA and AK could not be detected. Instead, cell
extracts contained an ADP-forming ACD activity of
0.2 U mg 1. The ACD activity in cells grown in the
absence of glucose was 0.022 U mg 1, indicating an
c. 10-fold induction of ACD upon growth on glucose.
Purification of ACD from C. aurantiacus and
identification of the encoding gene
ACD was purified from glucose-grown cells involving heat
precipitation followed by four chromatographic steps (Supporting Information, Table S1). In this procedure, ACD
was purified 507-fold to a specific activity of 45.6 U mg 1.
SDS-PAGE revealed one subunit with an apparent
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Published by John Wiley & Sons Ltd. All rights reserved
€nheit
M. Schmidt & P. Scho
174
(a)
(b)
Fig. 1. (a, b) Growth of Chloroflexus aurantiacus on 20 mM glucose, 0.1% yeast extract and 0.25% casamino acids (a) or on yeast extract/
casamino acids (b). The cultures were incubated under anaerobic conditions in the light. Growth (■), glucose consumption (●) and acetate
formation (▲) were followed over time.
molecular mass of 75 kDa (Fig. 3). The molecular mass of
the native ACD, determined by gel filtration on Superdex
200, was 300 kDa, indicating a homotetrameric structure.
The gene encoding ACD was determined by peptide mass
fingerprinting of the 75-kDa subunit, resulting in the identification of a single ORF, Caur_3920, in the genome of
C. aurantiacus. The matched peptides covered 42% of the
protein. Thus, Caur_3920 represents the ACD encoding
gene, acd, in C. aurantiacus. This was proved by functional
overexpression of Caur_3920 in E. coli.
Expression of the acd gene and purification of
recombinant ACD
Caur_3920 consisted of 2091-bp coding for a polypeptide
of 696 amino acids with a calculated molecular mass of
75.1 kDa. Caur_3920 was cloned into pET19b and transformed into E. coli BL21 (DE3)-codon plus RIL and
expressed. A 77-kDa His-tagged protein was produced in
accordance with the calculated molecular mass. The
recombinant ACD was purified to homogeneity by NiNTA affinity chromatography and gel filtration on Superdex 200 (Fig. 3b). The apparent molecular mass of
recombinant His-tagged ACD determined by gel filtration
was ca. 317 kDa. SDS-PAGE revealed one subunit of
77 kDa, indicating a homotetrameric structure of the
native enzyme.
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Published by John Wiley & Sons Ltd. All rights reserved
Catalytic properties and substrate specificity of
recombinant ACD
Kinetic properties of ACD catalyzing the reversible
conversion of acetyl-CoA/acetate (acetyl-CoA + ADP +
Pi ↔ acetate + ATP + CoA) were determined at 55 °C,
pH 7.5. In both directions of the reaction, the rate
dependence on the substrate concentrations followed
Michaelis–Menten kinetics. The apparent Vmax and Km
values in the direction of acetate formation were
51 U mg 1 and 0.037 mM (acetyl-CoA), 0.091 mM
(ADP), and 1.0 mM (phosphate), respectively. In the
direction of acetate activation the apparent Vmax and Km
values were 49 U mg 1, and 0.9 mM (acetate), 0.57 mM
(ATP), and 0.024 mM (HSCoA), respectively. The pH
optimum was at pH 7.5, with remaining activities of
about 40% at pH 7.3 and 59% at pH 7.8. ACD activity
was dependent on MgCl2, showing the highest activity at
about 5 mM. The temperature optimum was at 55 °C.
From the linear part of the Arrhenius plot between 20
and 55 °C, an activation energy of 49 kJ mol 1 was calculated. Different acyl- and aryl-acids were tested as substrates (10 mM) for ACD. Besides acetate (100%), the
ACD effectively utilized propionate (100%) and butyrate
(98%), thioglycolate (110%), and branched chain fatty
acids isovalerate (65%), isobutyrate (46%) and 2-methylbutyrate (75%). With lower activities, ACD accepted the
FEMS Microbiol Lett 349 (2013) 171–179
175
ADP-forming acetyl-CoA synthetase in Chloroflexus
This is the first report of a functional ACD involved in
acetate formation in the domain of Bacteria.
Acetate formation from glucose in
C. aurantiacus involves an ACD
Fig. 2. Glucose conversion to acetate in cell suspensions from
Chloroflexus aurantiacus. Glucose-grown cells were incubated under
anaerobic conditions in the dark in the presence of 10 mM glucose.
Glucose consumption (▲) and acetate formation (■) were followed
over time. As a control, acetate formation (●) was followed in the
absence of glucose.
aromatic acids imidazole-4-acetate (17%) and phenylacetate (10%). 4-Hydroxyphenylacetate, indol-3-acetate and
succinate were not utilized by ACD.
Culture and cell suspension experiments showed that
C. aurantiacus converted glucose to acetate as the major
fermentation product. Up to 1.5 mol acetate per mol glucose were formed. The formation of acetate by C. aurantiacus in glucose-containing media has been reported
earlier (Krasil’nikova & Kondrat’eva, 1987); however, acetate/glucose stoichiometries have not been determined.
Glucose degradation to pyruvate has been proposed to
involve the classical EM pathway, as concluded from
enzyme measurements and genome information (Kondratieva et al., 1992; Tang et al., 2011). However, the
enzymes involved in acetate formation have not been
analyzed. Here we report that in cell extracts of glucosegrown C. aurantiacus, activities of PTA and AK, that is
the enzymes typical for acetate-forming Bacteria, could
not be detected. This is in accordance with the absence of
the respective genes in the genome of the organism (Tang
et al., 2011). Instead, the cells contained an ADP-forming
ACD activity that was induced 10-fold in glucose-grown
cells, indicating a functional involvement in acetate formation from glucose. So far, ACDs have been reported in
prokaryotes to be typical for acetate-forming Archaea,
and this is the first report of the presence of ACD in a
bacterium. Thus, glucose degradation to acetate in the
bacterium C. aurantiacus appears to be a chimeric pathway consisting of a bacterial-like Embden–Meyerhof pathway, and an archaeal-like acetate-forming enzyme, ACD.
Discussion
The present study shows that the photoheterotrophic bacterium C. aurantiacus converted glucose to acetate as the
main product and that acetate formation from acetylCoA is catalyzed by an archael type ADP-forming ACD.
(a)
Caur-ACD is related to archaeal ACD I
isoenzymes
ACD from C. aurantiacus was purified and the encoding
gene acd was identified as Caur_3920, which was overex(b)
Fig. 3. (a, b) Purified ADP-forming ACD from
Chloroflexus aurantiacus (a) and recombinant
His-tagged enzyme isolated from transformed
Escherichia coli (b) as analyzed by SDS-PAGE.
(a) Lane 1, molecular mass markers, lane 2
purified enzyme after Mono-Q-Sepharose; (b)
Lane 1, molecular mass markers; lane 2,
purified recombinant enzyme.
FEMS Microbiol Lett 349 (2013) 171–179
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Published by John Wiley & Sons Ltd. All rights reserved
€nheit
M. Schmidt & P. Scho
176
Fig. 4. Comparison of subunit composition
and oligomeric structures of Chloroflexus
aurantiacus ACD and characterized archaeal
ACD I isoenzymes. Arrows indicate the ORFs
encoding the subunits and the subunit sizes.
Homologous regions of a-subunit are shaded
in dark gray and homologous regions of
b-subunit in light gray. Amino acid sequence
identities in comparison with the homologous
region of C. aurantiacus ACD are given as
percent.
Substrate
C. aurantiacus*
Direction of CoA-ester formation (%)
Acetate
100
Isobutyrate
91
Isovalerate
65
Indol-3-acetate
NM
Phenylacetate
10
Succinate
NM
Km-value (mM)
Acetate
0.90
ATP
0.57
HSCoA
0.02
Direction of acid formation (%)
Acetyl-CoA
100
Phenylacetyl-CoA
13
Isobutyryl-CoA
32
Km-value (mM)
Acetyl-CoA
0.037
ADP
0.09
1.0
KH2PO4
A. fulgidus†
H. marismortui‡
P. furiosus§
100
56
10
4
8
9
100
38
25
2
3
NM
100
85
ND
NM
NM
NM
0.34
0.133
0.027
100
NM
ND
0.01
0.01
0.11
1.7
0.38
0.06
100
1
ND
0.41
0.02
1.3
Table 1. Comparative analysis of substrate
specificities of recombinant Chloroflexus
aurantiacus ACD to characterized archaeal
ACD I isoenzymes
1.1
0.48
0.018
100
NM
32
0.025
0.15
0.396
Specific activities (U mg 1) are shown as percent activity in the direction of CoA-ester formation and acid formation, respectively. One hundred percent correspond to either 49 U mg 1
(acetate) or 51 U mg 1 (acetyl-CoA) (C. aurantiacus), 70 or 60 U mg 1 (Archaeoglobus fulgidus), 40 or 48 U mg 1 (Haloarcula marismortui) and 27 or 65 U mg 1 (Pyrococcus furiosus).
*Kinetic constants were determined at 55 °C as described in Materials and methods. Assay
mixture contained 10 mM of acid and 0.1 mM of CoA-ester.
†
€nheit (2002).
Musfeldt & Scho
‡
€nheit (2004b).
Br€
asen & Scho
§
Mai & Adams (1996).
NM, not measurable; ND, not determined.
pressed in E. coli to yield a recombinant ACD with similar properties as the native enzyme. Chloroflexus aurantiacus ACD, designated as Caur-ACD, was compared with
characterized ACDs from hyperthermophilic and halophilic Archaea with respect to molecular properties and
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Published by John Wiley & Sons Ltd. All rights reserved
substrate specificities. ACD from the hyperthermophiles
Pyrococcus furiosus and Thermococcus kodakaraensis comprise 145-kDa heterotetramers composed of two a-subunits (47 kDa) and two b-subunits (25 kDa) (Fig. 4; Mai &
Adams, 1996; Glasemacher et al., 1997; Shikata et al.,
FEMS Microbiol Lett 349 (2013) 171–179
177
ADP-forming acetyl-CoA synthetase in Chloroflexus
2007). The ACDs from the halophilic archaeon Haloarcula marismortui, and the hyperthermophilic Archaea
Archaeoglobus fulgidus and Methanococcus jannaschii are
145-kDa homodimeric proteins composed of subunits
representing fusions of homologous a- and b-subunits of
the P. furiosus ACD (Musfeldt & Sch€
onheit, 2002; Br€asen
& Sch€
onheit, 2004b). ACD from the eukaryotic protist
G. lamblia is composed of 78-kDa subunits (Sanchez
et al., 1999).
With respect to their substrate specificities, several
ACD isoenzymes have been identified in Archaea; for
example, in P. furiosus two ACD isoenzymes, I and II,
have been characterized. ACD I preferentially utilizes acetyl-CoA over aryl-CoA esters such as phenylacetyl-CoA
and is primarily involved in sugar fermentation, whereas
ACD II showed a preference for aryl-CoA esters over acetyl-CoA and is primarily implicated in the degradation of
(aromatic) amino acids (Mai & Adams, 1996; Glasemacher et al., 1997; Adams et al., 2001). ACD I and II enzymes
have also been characterized in A. fulgidus, an ACD II in
T. kodakaraensis and an ACD I in H. marismortui
(Musfeldt & Sch€
onheit, 2002; Br€asen & Sch€
onheit, 2004b;
Shikata et al., 2007). Besides ACD I and II, other ACD
isoenzymes with different substrate specificities have been
reported, for example, for Pyrobaculum aerophilum and
T. kodakaraensis and for the eukaryotic protist G. lamblia
(Sanchez & M€
uller, 1996; Br€asen & Sch€
onheit, 2004b;
Shikata et al., 2007).
Caur-ACD is composed of 75-kDa subunits which are
fusions of the a- and b-subunit homologs of P. furiosus
ACD. With a molecular mass of 300 kDa for the native
enzyme, Caur-ACD represents the largest of all ACDs
characterized so far. Based on substrate specificities for
CoA ester and the corresponding acids, Caur-ACD can be
attributed to ACD I isoenzymes from Archaea. A comparison of substrate specificities and kinetic constants of the
Caur-ACD and characterized archaeal ACD I isoenzymes
is given in Table 1. Caur-ACD and archaeal ACD I isoenzymes showed a strong preference for acetyl-CoA/acetate
over phenylacetyl-CoA/phenylacetate and accepted the
branched chain fatty acids isobutyrate and isovalerate, but
did not utilize succinate. These properties are characteristic of ACD I isoenzymes. Also, kinetic constants for
selected CoA esters and acids were similar in all ACD I
isoenzymes. ACD I of the anaerobic P. furiosus constitutes
a major energy-conserving site in sugar degradation (Mai
& Adams, 1996; Glasemacher et al., 1997; Adams et al.,
2001). In the aerobic H. marismortui, ACD I is proposed
to be involved in acetate formation from glucose as part
of an overflow mechanism (Br€asen & Sch€
onheit, 2004a).
41
Fig. 5. Phylogenetic relationships of
Chloroflexus aurantiacus ACD to selected ACD
of Archaea, Bacteria and Eukarya. The
numbers at the nodes are bootstrapping
values (%) according to maximum parsimony
(500 replicates). Only bootstrapping values
above 40% are shown. Alignment was
performed using Pyrococcus furiosus ACD I
a-subunit homologous region of the selected
ACDs. Analyses were conducted in MEGA 5.2.
Accession no.: Archaeoglobus fulgidus 1211
ACD I NP_070039.1; Chloroflexus aggregans
ZP_01517440.1; C. aurantiacus NC_010175;
Entamoeba histolytica XP_656290.1; Giardia
lamblia XP_001705744.1; Haloarcula
marismortui YP_135572.1; Haloferax volcanii
YP_003535057.1; Methanocaldococcus
jannaschii NP_247570.1; Methanosarcina
€1 NP_632382.1 Pelotomaculum
mazei Go
thermopropionicum YP_001212057.1;
Pyrobaculum aerophilum NP_560604;
P. furiosus ACD I NP_579269.1; P. furiosus
ACD II NP_578261.1; Roseiflexus castenholzii
YP_001430231.1; Syntrophus aciditrophicus
YP_460839.1; Thermococcus kodakaraensis
YP_183078.1.
FEMS Microbiol Lett 349 (2013) 171–179
G. lamblia
P. thermopropionicum
60
R. castenholzii
65
C. aggregans
99
100
C. aurantiacus
A. fulgidus 1211 ACDI
H. marismortui ACDI
46
100
H. volcanii
E. histolytica
M. jannaschii
S. aciditrophicus
74
P. aerophilum
M. mazei
P. furiosus ACDII
P. furiosus ACDI
67
100
T. kodakaraensis
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
€nheit
M. Schmidt & P. Scho
178
The similarity of Caur-ACD with ACD I isoenzymes of
Archaea is in accordance with its role in acetate formation from glucose in C. aurantiacus, reported in this
paper.
In summary, Caur-ACD represents the first functional
ACD within the domain of Bacteria. It is involved in the
formation of acetate from acetyl-CoA and thus likely substitutes for the classical bacterial acetate-forming enzymes
AK/PTA, which are missing in C. aurantiacus. It is proposed that Caur-ACD originates from archaeal ACD I
type isoenzymes, which are taken up by C. aurantiacus
via lateral gene transfer.
To identify the archaeal ACD I most closely related to
Caur-ACD, the degree of sequence identity, the subunit
size and the phylogenetic relationship of Caur-ACD were
analyzed in comparison with characterized archaeal ACD I
isoenzymes (Fig. 4). Caur-ACD showed similar high
sequence identity, about 45–50%, to all archaeal ACD I
isoenzymes. However, based on subunit size, Caur-ACD is
more closely related to ACD I isoenzymes from A. fulgidus
and H. marismortui, which are all composed of ‘fused’
70–85 kDa subunits. In contrast, P. furiosus ACD I consists of separate 25 kDa a- and 47 kDa b-subunits (Fig. 4).
In accordance with these findings, a phylogenetic tree of
Caur-ACD and selected bacterial homologs, characterized
archaeal ACD I enzymes and other ACDs mentioned in
text (Fig. 5), indicates that Caur-ACD is more closely
related to the archaeal ACD I enzymes from H. marismortui and A. fulgidus, rather than to ACD I from P. furiosus.
Thus, one might speculate that C. aurantiacus acquired its
ACD by lateral gene transfer of an ACD I isoenzyme from
Archaeoglobus/Haloarcula species. ACD homologs with
high sequence identity, about 50%, of Caur-ACD were
found in all Chloroflexaceae analyzed, suggesting a similar
function in acetate formation as in C. aurantiacus. Further, several syntrophic Bacteria such as S. aciditrophicus
and P. thermopropionicum, which form acetate in their
metabolism, do not contain AK/PTA genes (McInerney
et al., 2007; Kosaka et al., 2008) but do contain ACD
homologs. Thus, it is likely that these ACD homologs are
functionally involved in acetate formation, as shown for
C. aurantiacus. It should be noted, however, that the syntrophic bacterium Syntrophomonas wolfei does not contain
ACD homologs but instead AK/PTA genes, suggesting the
classical bacterial mechanism of acetate formation by AK/
PTA in this bacterium (Sieber et al., 2010).
Acknowledgements
We thank Melanie Brocker and Michael Bott (J€
ulich,
Germany) for MALDI-TOF MS analysis. This work was
supported by a grant from the Deutsche Forschungsgemeinschaft (SCHO 316/10-1).
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
References
Adams MW, Holden JF, Menon AL et al. (2001) Key role for
sulfur in peptide metabolism and in regulation of three
hydrogenases in the hyperthermophilic archaeon Pyrococcus
furiosus. J Bacteriol 183: 716–724.
Br€asen C & Sch€
onheit P (2004a) Regulation of acetate and
acetyl-CoA converting enzymes during growth on acetate
and/or glucose in the halophilic archaeon Haloarcula
marismortui. FEMS Microbiol Lett 241: 21–26.
Br€asen C & Sch€
onheit P (2004b) Unusual ADP-forming
acetyl-coenzyme A synthetases from the mesophilic
halophilic euryarchaeon Haloarcula marismortui and from
the hyperthermophilic crenarcheon Pyrobaculum aerophilum.
Arch Microbiol 182: 277–287.
Br€asen C, Schmidt M, Gr€
otzinger J & Sch€
onheit P (2008)
Reaction mechanism and structural model of ADP-forming
acetyl-CoA synthetase from hyperthermophilic Archaeon
Pyrococcus furiosus. J Biol Chem 283: 15409–15418.
Glasemacher J, Bock A-K, Schmid R & Sch€
onheit P (1997)
Purification and properties of acetyl-CoA synthetase
(ADP-forming), an archaeal enzyme of acetate formation
and ATP synthesis, from the hyperthermophile Pyrococcus
furiosus. Eur J Biochem 244: 561–567.
Herter S, Farfsing J, Gad’On N, Rieder C, Eisenreich W, Bacher
A & Fuchs G (2001) Autotrophic CO2 fixation by Chloroflexus
aurantiacus: study of glyoxylate formation and assimilation
via the 3-hydroxypropionate cycle. J Bacteriol 183: 4305–4316.
Kondratieva EN, Ivanovsky RN & Krasilnikova EN (1992)
Carbon metabolism in Chloroflexus aurantiacus. FEMS
Microbiol Lett 100: 269–272.
Kosaka T, Kato S, Shimoyama T, Ishii S, Abe T & Watanabe K
(2008) The genome of Pelotomaculum thermopropionicum
reveals niche-associated evolution in anaerobic microbiota.
Genome Res 18: 442–448.
Krasil’nikova EN & Kondrat’eva EN (1987) Growth of
Chloroflexus aurantiacus under anaerobic conditions in the
dark and the metabolism of organic substrates. Microbiology
56: 281–285.
Kunst A, Draeger B & Ziegenhorn J (1986) D-Glucose. Methods
of Enzymatic Analysis (Bergmeyer H-U, ed.), pp. 163–172.
Verlag Chemie, Weinheim.
Mai X & Adams MWW (1996) Purification and
characterization of two reversible and ADP-dependent acetyl
coenzyme A synthetases from the hyperthermophilic
archaeon Pyrococcus furiosus. J Bacteriol 178: 5897–5903.
McInerney MJ, Rohlin L, Mouttaki H et al. (2007) The
genome of Syntrophus aciditrophicus: life at the
thermodynamic limit of microbial growth. P Natl Acad Sci
USA 104: 7600–7605.
Musfeldt M & Sch€
onheit P (2002) Novel type of ADP-forming
acetyl coenzyme A synthetase in hyperthermophilic archaea:
heterologous expression and characterization of isoenzymes
from the sulfate reducer Archaeoglobus fulgidus and the
methanogen Methanococcus jannaschii. J Bacteriol 184:
636–644.
FEMS Microbiol Lett 349 (2013) 171–179
179
ADP-forming acetyl-CoA synthetase in Chloroflexus
Pierson BK & Castenholz RW (1974) A phototrophic gliding
filamentous bacterium of hot springs, Chloroflexus
aurantiacus, gen. and sp. nov. Arch Microbiol 100:
5–24.
Reeves RE, Warren LG, Susskind B & Lo HS (1977) An
energy-conserving pyruvate-to-acetate pathway in
Entamoeba histolytica. Pyruvate synthase and a new acetate
thiokinase. J Biol Chem 252: 726–731.
Sanchez LB & M€
uller M (1996) Purification and
characterization of the acetate forming enzyme, acetyl-CoA
synthetase (ADP-forming) from the amitochondriate protist,
Giardia lamblia. FEBS Lett 378: 240–244.
Sanchez LB, Morrison HG, Sogin ML & M€
uller M (1999)
Cloning and sequencing of an acetyl-CoA synthetase
(ADP-forming) gene from the amitochondriate protist,
Giardia lamblia. Gene 233: 225–231.
Sanchez LB, Galperin MY & M€
uller M (2000) Acetyl-CoA
synthetase from the amitochondriate eukaryote Giardia
lamblia belongs to the newly recognized superfamily of
acyl-CoA synthetases (nucleoside diphosphate-forming).
J Biol Chem 275: 5794–5803.
Sch€afer T, Selig M & Sch€
onheit P (1993) Acetyl-CoA
synthethase (ADP-forming) in archaea, a novel enzyme
involved in acetate and ATP synthesis. Arch Microbiol 159:
72–83.
Schaffer S, Weil B, Nguyen VD, Dongmann G, G€
unther K,
Nickolaus M, Hermann T & Bott M (2001) A
high-resolution reference map for cytoplasmic and
FEMS Microbiol Lett 349 (2013) 171–179
membrane-associated proteins of Corynebacterium
glutamicum. A high-resolution reference map for
cytoplasmic and membrane-associated proteins of
Corynebacterium glutamicum. Electrophoresis 22: 4404–4422.
Shikata K, Fukui T, Atomi H & Imanaka T (2007) A novel
ADP-forming succinyl-CoA synthetase in Thermococcus
kodakaraensis structurally related to the archaeal nucleoside
diphosphate-forming acetyl-CoA synthetases. J Biol Chem
282: 26963–26970.
Sieber J, Sims DR, Cliff H et al. (2010) The genome of
Syntrophomonas wolfei: new insights into syntrophic
metabolism and biohydrogen production. Environ Microbiol
12: 2289–2301.
Srere PA, Brazil H & Gonen L (1963) The citrate condensing
enzyme of pigeon breast muscle and moth flight muscle.
Acta Chem Scand 17: 129–134.
Tang KH, Barry K, Chertkov O et al. (2011) Complete genome
sequence of the filamentous anoxygenic phototrophic
bacterium Chloroflexus aurantiacus. BMC Genomics 12: 334.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Purification of ACD from Chloroflexus aurantiacus.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved