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Cloning, characterization and transcriptional analysis of twophosphate acetyltransferase isoforms from Azotobacter vinelandii
Maria Dimou • Anastasia Venieraki •
Georgios Liakopoulos • Panagiotis Katinakis
Received: 30 June 2010 / Accepted: 9 November 2010 / Published online: 21 November 2010
� Springer Science+Business Media B.V. 2010
Abstract Acetate is abundant in soil contributing to a
great extent on carbon cycling in nature. Phosphate ace-
tyltransferase (Pta, EC 2.3.1.8) catalyzes the reversible
transfer of the acetyl group from acetyl-P to CoA forming
acetyl-CoA and inorganic phosphate, participating to ace-
tate assimilation/dissimilation reactions. In the present
study, we demonstrate that Azotobacter vinelandii, a
nitrogen-fixing, free-living, soil bacterium, possesses two
class II phosphate acetyltransferase isoforms, AvPTA-1 and
AvPTA-2, with different kinetic properties. At the acetyl-
CoA forming direction, AvPTA-1 has lower affinity for
acetyl-P and higher affinity for CoA than AvPTA-2 while at
the acetyl-P forming direction; activity was measured only
for AvPTA-1. Quantification of their expression patterns by
RT-qPCR indicated that both genes are expressed during
exponential growth on glucose or acetate and are down-
regulated in the stationary phase. The ammonium avail-
ability during acetate growth resulted in up-regulation of
Avpta-2 expression only. Further, the gene expression
patterns of other related gene transcripts were also inves-
tigated, in order to understand the influence of each path-
way in the assimilation/dissimilation of acetate.
Keywords Acetate � Azotobacter vinelandii � Phosphate
acetyltransferase � RT-qPCR
Abbreviations
P Phosphate
CoA Coenzyme A
RT-qPCR Real-time quantitative polymerase chain
reaction
Acs-A Acetyl-CoA synthetase
Ack-A Acetate kinase
Pta Phosphate acetyltransferase
PoxB Pyruvate oxidase
Introduction
Acetate, a short-chain fatty acid, is abundant in soil and
other environments since it is a product of many fer-
mentative processes [1] and an important intermediate in
the anaerobic degradation of organic matter [2, 3]. Many
bacteria, including the non-symbiotically nitrogen-fixing
A. vinelandii, are able to use acetate as a source of carbon
and energy [4] while it represents the growth substrate for
methane producing archaea as well, contributing greatly
on carbon cycling in nature [5]. The first step in the uti-
lization of acetate is its activation into acetyl-CoA and
subsequently the operation of the glyoxylate cycle as an
anaplerotic pathway [6]. Once acetyl-CoA is made, it is
used to synthesize lipids, can enter the tricarboxylic acid
cycle to generate precursors of amino acids, or can be
oxidized to generate reducing power via diverse pathways
[7–10].
Two pathways are central to acetate catabolism in pro-
karyotes. Both of these pathways activate acetate into
acetyl-CoA, but they do it via different intermediates and in
response to different concentrations of acetate in the envi-
ronment. The first pathway is catalyzed by the acetyl-CoA
synthetase (Acs-A, EC 6.2.1.1) [11] while the second path-
way is comprised of the acetate kinase (Ack-A, EC 2.7.2.1)
M. Dimou � A. Venieraki � G. Liakopoulos � P. Katinakis (&)
Department of Agricultural Biotechnology, Agricultural
University of Athens, Iera Odos 75, 11855 Botanikos,
Athens, Greece
e-mail: [email protected]
123
Mol Biol Rep (2011) 38:3653–3663
DOI 10.1007/s11033-010-0478-3
and the phosphate acetyltransferase (Pta, EC 2.3.1.8) [12].
Although both pathways are reversible, hydrolysis of pyro-
phosphate generated by Acs-A prevents the Acs-A from
converting acetyl-CoA to acetate. The role of these path-
ways is to carefully maintain a physiological balance of
acetyl-CoA and free CoA [13–15]. On the contrary, acetate
formation occurs anaerobically during mixed acid fermen-
tation or aerobically when growth on excess glucose inhibits
respiration [15]. Two pathways are known to contribute to
acetate production: the first, assumed to be constitutive, is
the conversion of acetyl-CoA through phosphate acetyl-
transferase and acetate kinase [16, 17] while the second is
the conversion of pyruvate directly into acetate via pyruvate
oxidase (poxB, E.C 1.2.2.2) [18, 19].
Acetate/acetyl-CoA homeostasis is of great importance
to all cells and phosphate acetyltransferase which rapidly
converts acetyl-CoA via acetyl-P to acetate as an overflow
pathway or converts exogenous acetate back to acetyl-CoA
[16, 20], probably plays a central role in maintaining a
balance flux between biosynthesis and energy generation
[14]. Microbial genomes contain two main classes of
phosphate acetyltransferase enzymes [21] while an addi-
tional evolutionary distinct class also exists [22]. Class I
enzymes, which are *350 amino acids in length and are
not allosterically regulated, include enzymes from Clos-
tridium kluyverii [23], Veillonella alcalescens [24], Ther-
motoga maritime [25], Methanosarcina thermophila [26]
and Bacillus subtilis [27]. Class II enzymes, which are
*700 amino acids and allosterically regulated, with the
exception of enzymes from the enterobacteria Escherichia
coli [28, 29] and Salmonella enterica [21], remain less
investigated.
In the present study we present the enzymatic charac-
terization of two phosphate acetyltransferase class II iso-
forms from A. vinelandii. In addition, we examine the
transcript accumulation of acetate metabolism genes of
A. vinelandii under aerobic growth on glucose or acetate
minimal media. Since A. vinelandii is a nitrogen-fixing,
soil bacterium, we also investigate the influence of
ammonium source on the transcript accumulation of the
tested genes.
Materials and methods
Bacterial strains and growth conditions
E. coli XL-Blue1 strain (Invitrogen) was used for the
propagation of recombinant forms of the plasmid pET28a.
E. coli strain BL21 (DE3) (Novagen) was used for the
expression of recombinant proteins. All E. coli strains were
grown in LB medium supplemented with kanamycin.
A. vinelandii was grown at 30�C in LB medium or Burk’s
nitrogen-free salts [30], supplemented with either glucose
at 1% (BG) or sodium acetate at 0.3% (BA) and in Burk’s
salts supplemented with ammonium chloride at 0.1% and
either glucose at 1% (BNG) or sodium acetate at 0.3%
(BNA).
Heterologous expression of AvPTA-1 and AvPTA-2
in E. coli and purification of recombinant proteins
The coding sequences of Avpta-1 (YP_002800579) and
Avpta-2 (YP_002801221) were PCR-amplified from
A. vinelandii genomic DNA. The primers used were
AvPTA.1-F: 50-CCCCCATGGACACTCTATTTCTCGCC
CCCAC-30 with AvPTA.1-R: 50-AAACTCGAGGGCTCC
GACCGGCTGCCCCTG-30 and AvPTA.2-F: 50-CCCTCA
TGAAGACCTTCCTGATCGCCCCCAG-30 with AvPTA.
2-R: 50-AAAAAGCTTGCCGGCGGCGGTTTCGGCCT
G-30, carrying restriction sites for ligation to the pET28a
expression vector. The PCR-fragments were digested with
the appropriate restriction enzymes and cloned to the cor-
responding sites of pET28a, resulting in pET28a-AvPTA-1
and pET28a-AvPTA-2, respectively. The resulting con-
structs were confirmed by sequencing. Synthesis of
recombinant proteins was initiated by addition of 0.5 mM
isopropyl 1-thio-b-D-galactopyranoside when the cultures
reached A600 of 0.6 and continued cultivation for an
additional 4 h at 30�C. Cells were harvested by centrifu-
gation and were disrupted by sonication in Lysis buffer
(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole
supplemented with 1 mg/ml lysozyme). Cellular lysates
were centrifuged and the supernatants were used for pro-
tein purification. Recombinant proteins were purified with
Ni–NTA chromatography (Ni2?-nitrilotriacetate, Qiagen)
according to the manufacturer’s instructions. To remove
any imidazole and salts in the collected fractions, fractions
were dialysed against 50 mM Tris–HCl buffer (pH 7.2), for
12 h. The purity of the purified proteins was analyzed by
15% SDS-PAGE electrophoresis.
Phosphate acetyltransferase activity assay
The rate for both the forward (acetyl-CoA forming) and
reverse (acetyl-P forming) directions of the reaction cata-
lyzed by pta were measured at 35�C by monitoring the
change in absorbance at 233 nm concomitant with the
formation or the hydrolysis of the thioester bond of acetyl-
CoA (e = 4.36 M-1), using a 1 cm path-length quartz
cuvette in a HITACHI U-2800 spectrophotometer. The
standard reaction mixture (800 ll) for the forward direc-
tion contained 50 mM Tris–HCl (pH 7.2), 20 mM NH4Cl,
20 mM KCl, the appropriate substrate and a concentration
of enzyme sufficient to yield a linear rate over at least
2 min (approximately 3 lg). The standard reaction mixture
3654 Mol Biol Rep (2011) 38:3653–3663
123
(800 ll) for the reverse direction contained 50 mM Tris–
HCl (pH 7.2), 20 mM MgCl2, the appropriate substrate and
a concentration of enzyme sufficient to yield a linear rate
over at least 2 min (approximately 5 lg). Reactions were
initiated by addition of the second substrate as described
[31]. Kinetic parameters were estimated from non-linear
curve fitting using GraphPad Prism software (version 5 for
Windows, GraphPad Software). The kinetic values were
calculated by Michaelis–Menten or Substrate Inhibition
models. In the latter case, the equation used was:
Y = Vmax 9 X/[Km ? X 9 (1 ? X/Ki)], where Vmax is the
maximum enzyme velocity, if the substrate didn’t also
inhibit enzyme activity, Km is the Michaelis–Menten con-
stant, and Ki is the dissociation constant for substrate
binding in such a way that two substrates can bind to an
enzyme.
Size exclusion chromatography
Size exclusion chromatography was performed on a
Prominence liquid chromatography system (Shimadzu Co.
Tokyo, Japan) equipped with a ZORBAX GF-250 4 lm
column (4.6 9 250 mm, Agilent Technologies, Palo Alto,
CA, USA) and a SPD 20A diode array detector. Aliquots of
20 ll of protein samples were injected and eluted isocrat-
ically with 20 mM Na2HPO4 pH 7.0, 130 mM NaCl at a
flow rate of 1 ml/min. Molecular weight calibration was
performed by using bovine thyroglobulin (670 kDa),
bovine c-globulin (158 kDa), chicken ovalbumin (44 kDa),
horse myoglobin (17 kDa) and vitamin B12 (1.35 kDa)
mixture, all components of the Bio-Rad gel filtration
standard kit. Chromatograms were analyzed at 280 nm.
RNA isolation
Total RNA was isolated by the hot SDS/hot phenol method
[32]. Briefly, bacterial cultures were added to a 1/10 vol-
ume of 95% ethanol plus 5% saturated phenol to stabilize
cellular RNA and the cells were then harvested by centri-
fugation at 6,000 rpm for 5 min at 4�C. The supernatant
was aspirated and the pellets were kept at -80�C for no
longer than 1 week. Pellets were resuspended in 800 ll of
lysis buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA sup-
plemented with 3 mg/ml lysozyme) and 80 ll of a 10%
SDS solution was added to the lysate and incubated at 64�C
for 2 min. Following incubation, 88 ll of 1 M NaOAc (pH
5.2) was added to the lysate and an equal volume of water-
saturated phenol was added and incubated at 64�C for
6 min, inverting the tubes six times every 40 s. The sam-
ples were chilled on ice and centrifuged at 13,000 rpm for
10 min at 4�C. The aqueous layer was transferred to a tube
with equal volume of chloroform and centrifuged at
13,000 rpm for 5 min at 4�C. Subsequently, the aqueous
layer was ethanol precipitated by adding 1/10 volume of
3 M NaOAc (pH 5.2), 1 mM EDTA and two volumes cold
ethanol. The samples were incubated at -80�C overnight.
The RNA was pelleted by centrifugation at 13,000 rpm for
25 min at 4�C. Pellets were washed with ice cold 80%
ethanol and centrifuged at 13,000 rpm for 5 min at 4�C.
The pellets were resuspended in a total of 100 ll of RNase-
free water.
Total RNA was quantified using micro-spectrophotom-
etry (NanoDrop Technologies, Inc.) and its quality was
assessed by agarose gel electrophoresis. DNA was removed
with DNase I (Promega). Removal of DNA from the RNA
samples was confirmed by performing real-time PCR on
100 ng of total RNA using the Avin_38560-qPCR primer
set (Table 1), but without a reverse transcriptase step.
Those RNA samples found to yield Ct values larger than 35
were judged to be sufficiently free of contaminating DNA
for further analysis. Once more, total RNA was quantified
using micro-spectrophotometry (NanoDrop Technologies,
Inc.) while the A260/A280 ratio was always above two.
Purified RNA was either converted to cDNA immediately
or stored frozen at -80�C.
cDNA synthesis
First-strand cDNA synthesis was performed using Super-
script II RT (Invitrogen). Briefly, 1.5 lg of each RNA
sample was mixed with 1 ll of 250 ng/ll random hexamer
primers, 1 ll of 10 mM dNTP mix (Finnzymes), and
RNase-free water to make 12 ll. The mixture was
Table 1 Primers used in RT-qPCR reactions
Primer name Nucleotide sequence
Avin_34560-qPCR-F 50-AACTGGGCGCTGGTGCGTAT-30
Avin_34560-qPCR-R 50-TCCATGGCGGCGAGCATTTCA-30
Avin_41120-qPCR-F 50-TGGGCTGGAGCCTCACGGAAAT-30
Avin_41120-qPCR-R 50-CCAGGTTGCGCATGTCGTTGGA-30
Avin_34550-qPCR-F 50-CCGATACTGCTGGATCAGGT-30
Avin_34550-qPCR-R 50-CTGATAGCGGCTGACCACTT-30
Avin_41130-qPCR-F 50-GACTACTGTGCCAGTCACC-30
Avin_41130-qPCR-R 50-CCCGCTTTACCAGGTTGTAG-30
Avin_36780-qPCR-F 50-ATCCCTGCAGGCCCACCTCC-30
Avin_36780-qPCR-R 50-TGGCTATGGCGTGGTCCCGA-30
Avin_34530-qPCR-F 50-ATGTCGCGATGCGAGTGCCA-30
Avin_34530-qPCR-R 50-GCGTTGACCAATCCGGACACCT-30
Avin_42750-qPCR-F 50-GCAGCATCTACGGCGACCATCA-30
Avin_42750-qPCR-R 50-TGCGCCATCGCCGGTGAAAT-30
Avin_08480-qPCR-F 50-GGCCGGTGCTGATCGAGTTCAA-30
Avin_08480-qPCR-R 50-ACAGCAAGGTGCCGGCGAAT-30
Avin_38560-qPCR-F 50-TCCGGGTTGCCGAACATCACA-30
Avin_38560-qPCR-R 50-TGCGCAAGATCACCGGCAACA-30
Mol Biol Rep (2011) 38:3653–3663 3655
123
denatured at 65�C for 5 min and immediately chilled. 4 ll
of 59 first-strand buffer, 2 ll of 0.1 M DTT, 1 ll of
40 units/ll RNaseOUT (Invitrogen) and 1 ll of 200 units/
ll SuperScript II (Invitrogen) were then added and the
volume was adjusted to 20 ll. The mixture was incubated
at 25�C for 10 min, 42�C for 50 min, and finally 70�C for
15 min to inactivate the reaction.
RT-qPCR
The oligonucleotide primers used for RT-qPCR are listed
in Table 1. All primers were supplied by Invitrogen. The
primers were designed using Primer-BLAST (http://blast.
ncbi.nlm.nih.gov/Blast.cgi) and optimised to an equal
annealing temperature of 60�C. Each primer pair further
assessed for specificity with melting curve analysis and gel
electrophoresis of amplification products.
One microliter of diluted cDNA was added to the primer
pair mix and SYBR Green Master Mix (Applied Biosys-
tems) in each well. RT-qPCR was conducted on an MxPro
Mx3005P PCR system and analyzed with MxPro v4.01
software (Stratagene). The PCR cycling conditions were
95�C for 10 min, 40 cycles of 95�C for 30 s and 60�C for
1 min, 95�C for 1 min, 60�C for 30 s and 95�C for 30 s.
Plates were run for 40 cycles and fluorescence intensity
measured after every cycle. For each target sequence the
average cycle number at which fluorescence was detected
above background, in the exponential phase of amplifica-
tion, was obtained. No-reverse transcription controls were
included. The efficiency of each RT-qPCR reaction was
calculated using the LinRegPCR software [33].
Relative expression of the target gene in the various
Burk’s minimal media versus LB full medium was calcu-
lated using the following equation, described by Pfaffl [34].
Relative expression ratio per gene: R = EGOI^ (Ct, CONTROL
- Ct, SAMPLE) GOI/EREF^ (Ct, CONTROL-Ct, SAMPLE) REF. recA
(Avin_38560) was used as internal control. Statistical sig-
nificance was obtained using a pair-wise fixed reallocation
randomization test using the REST software [35].
Results
Avpta-1 and Avpta-2 encode putative phosphate
acetyltransferases
We identified three putative phosphate acetyltransferases
from A. vinelandii, AvPTA-1 (ACO79604), AvPTA-2
(ACO80246) and AvPTA-3 (ACO80523), all belonging to
the phosphate acetyl/butaryl transferase superfamily by
sequence homology searches using BLAST [36]. Microbial
genome sequencing has revealed two main classes of
phosphate acetyltransferase enzymes. Class I enzymes
are *350 amino acids in length, whereas class II enzymes
are *700 amino acids. Class I enzymes share end-to-end
homology with the C-terminal domain of class II enzymes;
hence it is inferred that the active site of class II enzymes is
located within their C-terminal domain [21]. AvPTA-1 (712
amino acids) and AvPTA-2 (691 amino acids) show 61.2%
identity between each other and 50.2 and 53% identity with
E. coli pta, respectively, all belonging to the Class II
enzymes. The N-terminal region of AvPTA-1 and AvPTA-2
contains a Bio-D and a DRTGG domain, as revealed by
CDD searches [37]. AvPTA-3 (317 amino acids), on the
other hand, has no additional domains, shows approxi-
mately 21% identity with E. coli eutD and only 8.9%
identity with E. coli pta, probably representing another
class of phosphate acetyl/butaryl transferases superfamily.
Figure 1 shows a ClustalW multiple protein alignment
among A. vinelandii AvPTA-1, AvPTA-2, AvPTA-3 and
other characterized phosphate acetyltransferases. Catalyti-
cally important Ser309, Arg310 and Asp316 residues (Met-
hanosarcina thermophila numbering) are all conserved in
AvPTA-1 and AvPTA-2 but not in AvPTA-3.
AvPTA-1 and AvPTA-2 exhibit phosphate
acetyltransferase activity
Using purified recombinant proteins, the catalytic proper-
ties of both AvPTA-1 and AvPTA-2 were investigated.
Both genes were PCR-amplified from A. vinelandii and
cloned to an expression vector to obtain the corresponding
His-tagged proteins, which were overproduced in E. coli
BL21 (DE3) cells and purified using Ni–NTA chroma-
tography. Analysis of the purified recombinant proteins by
SDS-PAGE and Coomassie blue staining, revealed single
protein bands, close to the theoretical molecular masses of
both proteins (77 and 76 kDa, respectively) (Fig. 2a, b). To
determine the biochemical characteristics of AvPTA-1 and
AvPTA-2, the kinetic constants were determined for both
directions of the reaction: acetyl-P ? CoA $ acetyl-
CoA ? Pi. The apparent kinetic constants of the substrates
were determined by varying the concentration of one
substrate around its Km value while leaving the other
unchanged at saturation concentrations.
Fig. 1 Sequence alignment of A. vinelandii AvPTA-1 (ACO79604)
and AvPTA-2 (ACO80246) and related enzymes. The sequences
included are Bacillus subtilis pta (CAB15793), Methanosarcinathermophila pta (AAA72041), Salmonella typhimurium pta
(AAL21239), E. coli pta (AAC75357), Lactobacillus sanfrancisensispta (BAB19267), E. coli eutD (AAC75511), Salmonella typhimuriumeutD (AAL21360) and A. vinelandii AvPTA-3 (ACO80523). Multiple
sequence alignment was performed using ClustalW [42]. Black boxesindicate identical amino acids while grey boxes indicate similar.
Black dots indicate amino acids involved in catalysis of Methano-sarcina thermophila pta [26]
c
3656 Mol Biol Rep (2011) 38:3653–3663
123
For the forward reaction (acetyl-CoA forming direction),
with acetyl-P as a variable substrate (Fig. 3a), AvPTA-1
showed substrate inhibition kinetics (Ki = 5.2 mM) with
Km = 1 ± 0.4 mM and Vmax = 0.1 ± 0.02 lmol s-1 lg-1.
With CoA as a variable substrate (Fig. 3b), AvPTA-1 showed
inhibition kinetics (Ki = 0.1 mM) with Km = 0.2 ± 0.1
mM and Vmax = 0.1 ± 0.06 lmol s-1 lg-1. These charac-
teristics are within the range of other bacterial and archaea
species (Brenda, http://www.brenda-enzymes.org/).
For the reverse reaction (acetyl-P forming direction)
with acetyl-CoA as a variable substrate (Fig. 3c), AvPTA-1
showed Michaelis–Menten kinetics with Km = 0.05 ±
0.01 mM and Vmax = 0.003 ± 0.001 lmol s-1 lg-1, while
with phosphate as a variable substrate (Fig. 3d), AvPTA-1
showed inhibition kinetics (Ki = 19 mM) with Km = 11 ±
9 mM and Vmax = 0.004 ± 0.002 lmol s-1 lg-1. These
characteristics, as well, are within the range of other bac-
terial and archaea species (Brenda, http://www.brenda-
enzymes.org/).
AvPTA-2, on the other hand, for the forward reaction
(acetyl-CoA forming direction), with acetyl-P as a variable
substrate (Fig. 4a), showed substrate inhibition kinetics
(Ki = 12 mM) with Km = 0.5 ± 0.1 mM and Vmax =
0.004 ± 0.0004 lmol s-1 lg-1. With CoA as a variable
substrate (Fig. 4b), AvPTA-2 showed inhibition kinetics
(Ki = 0.04 mM) with Km = 0.3 ± 0.1 mM and Vmax =
0.04 ± 0.02 lmol s-1 lg-1, as well within the range of
other species (Brenda, http://www.brenda-enzymes.org/).
For the reverse reaction (acetyl-P forming direction) we
could not detect any enzyme activity for AvPTA-2 under
our experimental conditions.
Oligomeric state analysis of AvPTA-1 and AvPTA-2
In order to gain an insight into the oligomeric state of
recombinant AvPTA-1 and AvPTA-2, we performed gel fil-
tration analysis. The recombinant AvPTA-1 was estimated as
a *163 kDa peak, probably representing a dimer, since the
theoretical molecular mass of AvPTA-1 is *77 kDa. Simi-
larly, recombinant AvPTA-2 was estimated as a *165 kDa
peak, probably representing a dimer as well, since the the-
oretical molecular mass of AvPTA-2, is *76 kDa.
A. vinelandii’s acetate metabolism under various
physiological and growth conditions
The effect of growth phase (exponentially or stationary
grown cells), carbon source (acetate or glucose) and source
of fixed nitrogen (ammonium supplied either by nitrogen
fixation or exogenously supplied to the medium) on the
accumulation of gene transcripts involved in acetate
assimilation or dissimilation was examined by real-time
RT-qPCR. Cells from the exponential and the stationary
phases were harvested and the relative gene expression
levels were calculated against rich LB medium grown cells
from the same growth phases.
Figure 5 presented a simplified model of the acetate
metabolism pathways examined in the present study. We
suppose that acetyl-CoA can be metabolized into acetate by
the phosphate acetyltransferase (pta, E.C 2.2.1.8,
Avin_34550, Avin_41130) and acetate kinase (Ack-A, E.C
2.7.2.1, Avin_34560, Avin_41120) pathway. An acylphos-
phatase (AcPh, E.C 3.6.1.7, Avin_36780) may possibly
convert acetyl-P into acetate while the acetyl-Co/acetate
interconversion could also be made by the acetyl-CoA syn-
thetase (Acs-A, E.C 6.2.1.1, Avin_34530, Avin_42750).
Moreover, a thiamine diphosphate-dependent pyruvate
dehydrogenase (poxB, E.C 1.2.2.2, Avin_08480) can pro-
duce acetyl-P from pyruvate.
When A. vinelandii was exponentially grown on glucose
minimal medium as the primary carbon source, Avacs-A1
transcript levels were elevated (P \ 0.05) (Fig. 6c) while
Avpta-1 and Avpta-2 were stably expressed (Fig. 6a, b).
Growth on acetate minimal medium, on the other hand,
Fig. 2 SDS-PAGE analysis of expression and purification of
recombinant AvPTA-1 and AvPTA-2. a SDS-PAGE analysis of the
soluble cytoplasmic fraction from E. coli BL21 (DE3) cells
overexpressing AvPTA-1 lane 1, and the elution fraction after the
Ni–NTA purification of the recombinant AvPTA-1 lane 2. b SDS-
PAGE analysis of the soluble cytoplasmic fraction from E. coli BL21
(DE3) cells overexpressing AvPTA-2 lane 1, and the elution fraction
after the Ni–NTA purification of the recombinant AvPTA-2 lane 2
3658 Mol Biol Rep (2011) 38:3653–3663
123
decreased Avacs-A1 expression (P \ 0.05) (Fig. 6c) while
Avpta-1 and Avpta-2 remained stably expressed although
they showed increased expression compared to the glucose
growth (P \ 0.05, Anova) (Fig. 6a, b). The presence of
ammonium in the glucose minimal medium down-regu-
lated Avacs-A1 expression (P \ 0.05) (Fig. 6c) while its
presence in the acetate minimal medium up-regulated
Avpta-2 expression (P \ 0.05) (Fig. 6b). Avacs-A2
retained its basal levels of expression during exponential
growth in all conditions tested (Fig. 6d).
During A. vinelandii stationary phase growth on either
glucose or acetate minimal media most of the studied genes
showed a tendency for down-regulation (Fig. 6). However,
during the stationary phase, Avacs-A1 retained its basal level
Fig. 3 Phosphate acetyltransferase activity of the forward (acetyl-
CoA forming direction) and reverse reaction (acetyl-P forming
direction) catalysed by AvPTA-1. a Acetyl-CoA forming direction.
The CoA was kept constant at 0.125 mM, while the acetyl-P
concentration was varied. b Acetyl-CoA forming direction. The
acetyl-P was kept constant at 2.5 mM, while the CoA concentration
was varied. c Acetyl-P forming direction. The phosphate was kept
constant at 12.5 mM, while the acetyl-CoA concentration was varied.
d Acetyl-P forming direction. The acetyl-CoA was kept constant at
0.1 mM, while the phosphate concentration was varied. Mean values
were obtained from three independent replicates and error barsrepresent standard errors. Non-linear regression analysis was used in
order to fit the data to curves using GraphPad Prism v5.0 (GraphPad
Software, San Diego, CA)
Fig. 4 Phosphate acetyltransferase activity of the forward (acetyl-
CoA forming direction) catalysed by AvPTA-2. a Acetyl-CoA
forming direction. The CoA was kept constant at 0.025 mM, while
the acetyl-P concentration was varied. b Acetyl-CoA forming
direction. The acetyl-P was kept constant at 2 mM, while the CoA
concentration was varied. Mean values were obtained from three
independent replicates and error bars represent standard errors. Non-
linear regression analysis was used in order to fit the data to curves
using GraphPad Prism v5.0 (GraphPad Software, San Diego, CA)
Mol Biol Rep (2011) 38:3653–3663 3659
123
of expression when ammonium was present in the acetate
minimal medium (Fig. 6c) while Avacs-A2 retained its basal
level of expression in the acetate minimal medium (Fig. 6d).
Finally, we could not detect either AvAcPh or AvpoxB
expression under these growth and physiological
conditions.
Discussion
Acetate is abundant in soil and other environments as a
product of many fermentative and degradative processes
[1–3] and since it constitutes the growth substrate for
methane producing archaea as well, it represents a major
intermediate in the global carbon cycle [5]. Phosphate
acetyltransferase rapidly converts acetyl-CoA via acetyl-P
to acetate as an overflow pathway or converts exogenous
acetate back to acetyl-CoA [14]. Two main classes of
phosphate acetyltransferase enzymes have been identified
with class I enzymes to be *350 amino acids in length,
whereas class II enzymes *700 amino acids [21].
AvPTA-1 and AvPTA-2 from the soil, nitrogen-fixing
A. vinelandii, belong to the Class II enzymes, with their
N-terminal regions containing Bio-D and DRTGG domains
Fig. 5 Simplified model for the acetate metabolism pathways
examined in the present study. Pta phosphate acetyltransferase,
Ack-A acetate kinase, AcPh acylphosphatase, Acs-A acetyl-CoA
synthetase, poxB thiamine diphosphate-dependent pyruvate
dehydrogenase
Fig. 6 Relative expression levels of Avpta-1 (a) Avpta-2 (b) Avacs-
A1 (c) and Avacs-A2 (d) in A. vinelandii cultures grown in various
Burk’s minimal media (BG Burk’s with glucose, BGN Burk’s with
glucose and ammonium, BA Burk’s with acetate, BAN Burk’s with
acetate and ammonium) versus A. vinelandii cultures grown in LB
full medium as determined by RT-qPCR. Fold change was calculated
according to the equation described in the Materials and methods with
normalization against recA. The data are the mean of three biological
replications while the bars represent standard errors. Up-pointingarrow indicate up-regulation (P \ 0.05), down-pointing arrow indi-
cate down-regulation (P \ 0.05) and rectangular indicate not statis-
tically significant regulation (P [ 0.05), calculated with the REST
software [35]
3660 Mol Biol Rep (2011) 38:3653–3663
123
while their C-terminal domains correspond to the pta
domain.
Our data demonstrate that both AvPTA-1 and AvPTA-2
show phosphate acetyltransferase activity in the acetyl-
CoA forming direction while for the acetyl-P forming
direction we were able to measure enzyme activity only for
the AvPTA-1 isoform. AvPTA-1 activity is greater in the
acetyl-CoA forming direction (Vmax = 0.1 ± 0.02 l-mol s-1 lg-1) than the acetyl-P forming direction
(Vmax = 0.003 ± 0.001 lmol s-1 lg-1) while AvPTA-2
activity in the acetyl-CoA forming direction was lower
than the AvPTA-1 activity at the same direction
(Vmax = 0.004 ± 0.0004 vs. 0.1 ± 0.02 lmol s-1 lg-1).
These data taken together suggest that AvPTA-1 has lower
affinity to acetyl-P, as shown by a Km for acetyl-P that is
approximately one order of magnitude higher than that of
AvPTA-2 (1 ± 0.4 vs. 0.5 ± 0.1 mM). In contrast,
AvPTA-1 has higher affinity than the AvPTA-2 to CoA
(0.2 ± 0.1 vs. 0.3 ± 0.1 mM). The different enzyme
kinetics of the isoforms suggest that the pta gene products
may serve different biological roles under certain
metabolic conditions which are imposed by the availabil-
ity of carbon sources and/or maintenance of acetyl-P
concentration.
In most bacteria, pta and ack-A genes are organized into
a single operon and they are involved in the maintenance of
the intracellular acetyl-CoA and acetyl-P pools [38].
However, the conversion of acetyl-CoA to acetate and ATP
often does not go to completion so cells maintain a sig-
nificant pool of acetyl-P, which serves as a storage mole-
cule of carbon, phosphate and energy as well as a global
signal [39]. Our biochemical and in silico analyses revealed
that A. vinelandi genome contains two functional phos-
phate acetyltransferase isoforms as well as two putative
acetate kinases and acetyl-CoA synthetases. Orthologs
neighbourhood analyses revealed that in A. vinelandi the
genes coding for these enzymes are organized into two pta-
ack-A operons, Avpta-1-Avack-A1 and Avpta-2-Avack-A2.
Furthermore, Avacs-A1 is also adjacent to the former
operon while Avacs-A2 is distant from the latter operon.
Our results show that the two phosphate acetyltransfer-
ase genes are constitutively expressed during the expo-
nential growth and the encoded enzymes show different
kinetic constants for the acetate catabolism as well as the
acetate production routes. So the acetate assimilation/dis-
similation system could function under a wide range of
acetate concentrations allowing A. vinelandii to adapt and
survive under the prevailing and/or changing environ-
mental conditions. Further, the allosteric regulation of the
Class II PTA enzymes by pyruvate, phosphoenolpyruvate,
NADH and ATP [21, 29, 40] indicates the central role
of that enzyme in the carbon and energy status of the
cell. Moreover, deletion of the E. coli phosphate
acetyltransferase resulted in increased sensitivity to envi-
ronmental changes and de-regulation of the central
metabolism [41]. Nevertheless, the existence of paralogs
for phosphate acetyltransferase, acetate kinase and acetyl-
CoA synthetase in A. vinelandii adds an extra level of
complexity in acetate metabolism of this soil bacterium.
A. vinelandii is among the few bacterial species able to
fix atmospheric nitrogen under aerobic conditions. Thus,
investigation of the expression pattern of genes involved in
acetate metabolism under conditions where ammonium is
either exogenously supplied or produced via nitrogen fix-
ation would provide valuable information as to the survival
and growth of these bacterial species in the soil environ-
ment. Our data demonstrated that when A. vinelandii was
exponentially grown under nitrogen-fixing conditions, on
glucose minimal medium as the primary carbon source,
Avacs-A1 transcript levels were elevated while Avpta-1 and
Avpta-2 were stably expressed. Growth on acetate minimal
medium, on the other hand, decreased Avacs-A1 expression
while Avpta-1 and Avpta-2 remained stably expressed
although they showed increased expression compared to
the glucose growth. These data taken together suggest that
rapidly growing and nitrogen-fixing A. vinelandii uses the
pta pathway when acetate is available, and the acs-A
pathway when glucose is available as a carbon source. The
presence of fixed nitrogen (ammonium) in the glucose
minimal medium down-regulated Avacs-A1 expression
while its presence in the acetate minimal medium up-reg-
ulated Avpta-2 expression indicating that the availability of
fixed nitrogen in combination with available carbon source
determines whether the acs-A or the pta pathway is used
for acetate assimilation.
During A. vinelandii stationary phase growth under
nitrogen-fixing conditions, on either glucose or acetate
minimal media, most of the studied genes showed a ten-
dency for down-regulation. However, during the stationary
phase, Avacs-A1 retained its basal level of expression when
ammonium was present in the acetate minimal medium
while Avacs-A2 retained its basal level of expression in the
acetate minimal medium, indicating the critical role of the
acs-A acetate assimilation system during growth on
acetate.
In conclusion, we provide evidence that A. vinelandii
genome contains two functional phosphate acetyltransfer-
ase isoforms as well as two differentially transcribed
acetyl-CoA synthetase isoforms. The co-expression of both
Avpta genes in combination with the homo-dimeric form of
both enzymes raises questions whether or not hetero-
dimeric PTA enzymes are formed in vivo and whether they
display any enzyme activity. Further studies on the func-
tional characterization of these enzymes by inactivation of
either of the genes or both genes would better clarify their
specific roles during acetate metabolism.
Mol Biol Rep (2011) 38:3653–3663 3661
123
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