JB Accepts, published online ahead of print on 15 September 2006 J. Bacteriol. doi:10.1128/JB.00710-06 Copyright © 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Pirin regulates pyruvate catabolism through interaction with the pyruvate dehydrogenase E1 subunit and modulating pyruvate dehydrogenase activity
Running Title: Pirin regulates pyruvate dehydrogenase activity
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Po-Chi Soo,1,a Yu-Tze Horng,1,a Meng-Jiun Lai,3 Jun-Rong Wei,1 Shang-Chen Hsieh,1 Yung-Lin Chang,1 Yu-Huan Tsai1and Hsin-Chih Lai1,2,*
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1
Department of Clinical Laboratory Sciences and Medical Biotechnology, National
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Taiwan University College of Medicine, Taipei, Taiwan, R.O.C. 2
Department of Laboratory Medicine, National Taiwan University Hospital and
National Taiwan University College of Medicine, Taipei, Taiwan, R.O.C.
C A 3
Department of Laboratory Medicine and Biotechnology, College of Medicine, Tzu
Chi University, Hualien, Taiwan
a
The two authors contributed equally to this work.
*Correspondence: Dr. Hsin-Chih Lai, Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, No. 1 Chan-Der Street, Taipei 100, Taiwan (R.O.C.).
Tel. +886 2 2312 3456 (ext 6931), Fax +886 2 2371 1574 E-mail:
[email protected]
1
1 2
Abstract The protein Pirin, which is involved in a variety of biological processes, is
3
conserved from prokaryotic micro-organisms, fungi, and plants to mammals. It acts as
4
a transcriptional cofactor or an apoptosis-related protein in mammals, and involves
5
seed germination and seedling development in plants. In prokaryotes, while Pirin is
6
stress-induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the
7
functions of Pirin orthologs remain mostly uncharacterized. We show that the Serratia
8
marcescens pirinSm gene encodes an ortholog of Pirin protein. Protein pull-down and
9
bacterial two-hybrid assays followed by SDS-PAGE and electrospray ionization (ESI)
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MASS-MASS analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a
11
component interacting with PirinSm. Functional analyses showed that both PDH E1
12
subunit activity and PDH enzyme complex activity are inhibited by PirinSm in S.
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13
marcescens CH-1. The S. marcescens CH-1 pirinSm gene was subsequently mutated
14
by insertion-deletion homologous recombination. Accordingly, the PDH E1, PDH
15
enzyme complex activities and cellular ATP concentration increased up to 250%,
16
140%, and 220%, respectively, in the S. marcescens CH-1 pirinSm mutant.
17
Concomitantly, the cellular NADH/NAD+ ratio increased in the pirinSm mutant,
18
indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that
19
PirinSm plays a regulatory role in the process of pyruvate catabolism to acetyl-CoA
20
through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex
21
activity in S. marcescens CH-1, and suggest PirinSm is an important protein involved
22
in determining the direction of pyruvate metabolism to go towards either the TCA
23
cycle or the fermentation pathways.
2
1
INTRODUCTION
2
The protein Pirin is widely found in mammals, plants, fungi and also
3
prokaryotic organisms (32). While the cellular functions of Pirin show diversity and
4
Pirin homologues play important roles in a number of different biological processes,
5
cellular localization of Pirin is not restricted to specific compartments. In eukaryotes,
6
Pirin was initially isolated through a yeast two-hybrid screen from the Hela cell
7
cDNA library and is localized within cell nuclei; it acts as an interactor of nuclear
8
factor I/CCAAT box transcription factor (NFI/CTF1) (32). In an attempt to identify
9
downstream nuclear targets of Bcl-3 using a yeast two-hybrid screening for the
10
expression cDNA library derived from human activated B cells, Pirin interacts with
11
and increases the DNA-binding activity of Bcl-3-p50 complex (Bcl-3 is a member of
12
the IκB family that inhibits NF-κB activity) (8). A recent report from Orzaez et al.,
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(2001) further showed that lepirin, a tomato homologue of human Pirin, is involved in
14
programmed cell death (21). On the other hand, in Arabidopsis, a Pirin ortholog
15
(AtPirin1) was isolated as a protein interacting with the α-subunit of G protein
16
through the yeast two-hybrid screen. AtPirin1 is involved in the regulation of seed
17
germination and early seedling development of Arabidopsis (16). The human Pirin
18
crystalline structure was subsequently determined by Pang et al., (2004), who showed
19
that Pirin comprises two β-barrel domains, with a potential Fe(II) cofactor bound
20
within the cavity of the N-terminal domain. These findings suggest an enzymatic role
21
for Pirin, most likely in biological redox reactions involving oxygen (22).
22
In prokaryotes, pirA encoding an ortholog of Pirin together with an adjacent gene,
23
pirB, is up-regulated under high salinity and some other stress conditions in
24
cyanobacterium Synechocystis sp. PCC 6803 (13). However, induction of the pirAB
3
1
genes is not related to programmed cell death, and disruption of pirA did not affect the
2
cellular gene expression profile (13). Adams and Jia (2005) determined the crystalline
3
structure of the Pirin homologue YhhW from Escherichia coli, in which the YhhW
4
structure shows similarity to human Pirin. Through active site analysis, YhhW was
5
further shown to act as a 2,3-dioxygenase that degrades the antioxidant quercetin to 2-
6
protocatechuoylphloroglucinol, with concomitant release of CO as a byproduct (1).
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7
Previous studies on selecting for precocious-swarming mutants derived from
8
Serratia marcescens CH-1 by transposon mutagenesis (15, 28) identified a mutant
9
strain in which a pirin gene homologue was inserted by a mini-Tn5 transposon (Soo
10
and Lai, unpublished data). In comparison to human Pirin, which is a 32-kDa protein
11
consisting of 290 amino acids and E. coli Pirin (YhhW), which is a 25.4-kDa protein
12
with 231 amino acids, bioinformatics analyses identified a 312-amino acid, 35-kDa
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13
Pirin
ortholog
of
S.
marcescens
14
http://www.sanger.ac.uk/cgi-bin/blast/submitblast/s_marcescens). Subsequently, a 5-
15
kb pirinSm locus was cloned and sequenced in S. marcescens strain CH-1. In this
16
communication, using the protein pull-down and bacterial two-hybrid screening
17
assays followed by protein identification by electrospray ionization (ESI) MASS-
18
MASS analyses, we showed that the Pirin ortholog (PirinSm) in S. marcescens CH-1
19
interacts with the E1 subunit of pyruvate dehydrogenase (PDH) complex. PDH E1 is
20
one of the three subunits (E1, pyruvate dehydrogenase; E2, dihydrolipoamide
21
dehydrogenase transacetylase; E3, lipoamide dehydrogenase) of the PDH multi-
22
enzyme complex, which is an assemblage that plays a pivotal role in cellular
23
carbohydrate metabolism, catalyzing the oxidative decarboxylation of pyruvate and
24
the subsequent acetylation of coenzyme A (CoA) to form acetyl-CoA (5, 19). During
4
strain
Db11
(Sanger
Institute,
1
the process of PDH enzyme complex reactions, PDH E1 is responsible for the first
2
step of the multi-step process and catalyses pyruvate decarboxylation, followed by
3
transferring the hydroxyethyl group to thiamin diphosphate (ThDP) that together with
4
Mg2+ acts as the reaction cofactor (6). Subsequent gene deletion and biochemical
5
analyses showed that PirinSm regulated (inhibited) PDH E1 and PDH enzyme complex
6
activities. In accordance, the cellular ATP concentration and ratio of NADH/NAD+
7
increased in the pirinSm-deleted S. marcescens mutant grown to late logarithmic phase.
8
These results show a new role of PirinSm involving in the regulation of pyruvate
9
catabolism to acetyl-CoA. This may subsequently affect cellular central carbohydrate
10
metabolism to go towards the tricarboxylic acids (TCA) cycle or fermentation
11
pathways.
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MATERIALS AND METHODS Bacterial strains, plasmids, primers and culture conditions. S. marcescens
4
CH-1 (28) is a clinical isolate routinely maintained at 37°C on Luria Bertani (LB)
5
plate. The chromosomal DNA sequence of S. marcescens Db11 was determined at the
6
Sanger Institute (http://www.sanger.ac.uk/cgi-bin/blast/submitblast /s_marcescens).
7
The bacterial strains, plasmids and primers used in this study are described in Table 1.
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Enzymes and chemicals. DNA restriction and modification enzymes were
10
purchased from Roche (Mannheim, Germany). Taq polymerase and PCR-related
11
products were obtained from either Perkin Elmer (Boston, MA, USA) or Takara
12
Biomedicals (Shiga, Japan). Other laboratory grade chemicals were purchased from
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Sigma Chemical Company (St. Louis, MO, USA), Merck (Schwabach, Germany).
15
Recombinant DNA techniques. Unless otherwise indicated, standard protocols
16
were used for DNA/DNA hybridization, plasmid and chromosomal DNA preparation,
17
transformation, electroporation, PCR, restriction digestion, agarose gel electrophoresis,
18
DNA recovery from agarose gels, DNA ligation, and conjugation. Southern blotting
19
analysis of chromosomal DNA was performed using nylon membranes (Hybond N+;
20
Amersham, Piscataway, NJ, USA) and a DIG High Prime labeling kit (Roche)
21
according to the recommendations of the manufacturer. PCR DNA amplicons were
22
cloned using the pCR 2.1 and the TA Cloning Kit (Invitrogen, Carlsbad, CA, USA).
23
DNA sequencing and analysis were performed using a Perkin-Elmer Autosequencer
24
model 377 with a Taq DyeDeoxy terminator cycle sequencing kit (Applied
6
1
Biosystems, Foster, CA, USA). The DNA sequences of PCR products were confirmed
2
by sequencing both strands from two or three independent reactions.
3 4
Cloning the pirinSm locus in S. marcescens CH-1. The primers Db11-pir-F/
5
Db11-pir-R (Table 1) designed from S. marcescens Db11 pirin DNA sequence were
6
used to amplify a 924-bp partial pirin DNA fragment showing high nucleic acid
7
sequence identity (99%) to Db11 pirin from S. marcescens strain CH-1. This DNA
8
fragment was subsequently labeled and used as a probe to clone the pirinSm locus from
9
S. marcescens CH-1. Conventional restriction digestions, Southern blot hybridizations,
10
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cloning and sequencing identified a 5-kb DNA fragment containing pirinSm (Fig. 1).
11 12
Construction of S. marcescens CH-1 pirinSm insertion-deletion mutant S.
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13
marcescens PC103. For construction of the pirinSm mutant, a protocol was designed
14
for specific insertion of a 2-kb streptomycin (Sm)-resistant Ω cassette, excised from
15
pHP45Ω, into the pirinSm gene in S. marcescens CH-1 (23, 28). Briefly, the 5'-region
16
of pirinSm was amplified by PCR using primer pair Pirk1 and Pirk2, TA-cloned into
17
pCR 2.1 (Invitrogen) and excised as a SalI/HindIII fragment. A second PCR product
18
encompassing the 3'-region of pirinSm was generated using primer pair Pirk3 and
19
Pirk4, TA-cloned into pCR 2.1 (Invitrogen) and excised as a HindIII/EcoRI fragment.
20
The two DNA fragments together with the Ω cassette were ligated with the
21
SalI/EcoRI digested pUT-mini-Tn5-Km1 suicide vector (7) to form plasmid pUT-
22
pirinSm::Sm.
23
For gene inactivation by homologous recombination, pUT-pirinSm::Sm was
24
transferred from E. coli S17-1 λ pir to S. marcescens CH-1 by conjugation (15).
7
1
Transconjugants were spread on LB plates containing streptomycin (100 µg/ml) and
2
tetracycline (13 µg/ml). Mutant candidates were screened by colony PCR. Southern
3
blot hybridization using the pirinSm gene as a probe was performed to confirm the
4
mutant genotype in which a double-crossover event had occurred (data not shown).
5
The resultant pirinSm mutant strain was designated as S. marcescens PC103.
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Western blot analysis. The Western blot procedures were modified from
8
Sambrook et al. (24). In brief, bacterial cells harvested were washed once in PBS (pH
9
7.5) and resuspended in cell lysis buffer (20 mM PIPES, pH 7.2; 100 mM NaCl; 1
10
mM PMSF). Samples were left for 30 min on ice and centrifuged at 14000 rpm for 30
11
min at 4oC. The spent supernatants were then concentrated and analyzed in 12.5%
12
SDS-PAGE. Proteins separated were transferred to PVDF membrane (Amersham)
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and incubated in blocking buffer (5% in milk, 0.1% Tween 20) for 1 h. Further
14
incubations with anti-His monoclonal antibody or anti-PirinSm polyclonal antibodies
15
were then performed in blocking buffer for 1 h at room temperature, followed by
16
addition of horse radish peroxidase-conjugated anti-rabbit second antibody for another
17
1 h before development and X-ray film exposure (28).
18 19
GST pull-down assay. The ProFound Pull-Down GST Protein: Protein
20
Interaction Kit (PIERCE, Rockford, IL, USA) was used. Over-synthesis of GST
21
fusion proteins was achieved by culturing E. coli DH5α cells containing recombinant
22
plasmids pSC10 (GST tagged PirinSm fusion protein in pGEX) in 3 ml of LB broth
23
medium to the mid-logarithmic phase at 37°C, followed by addition of IPTG at the
24
final concentration of 0.5 mM for induction. After further culture for 3 h, cells were
8
1
centrifuged and then suspended in 100 µl of lysis buffer (20 mM Tris-HCl, pH 8.0;
2
100 mM NaCl; 1 mM EDTA; 0.5% NP-40) containing leupeptin (1 µg/ml), pepstatin
3
A (1 µg/ml) and phenylmethylsulfonyl fluoride (1 mM). Glutathione-sepharose 4B
4
beads (20 µl) (Amersham) were then added to the spent supernatant and the mixture
5
was incubated under mild shaking for 2 h at 4°C. Beads washed three times with
6
PBST buffer (PBS, pH 7.4; 1% Triton X-100) were subsequently added to 500 µl of
7
spent cell lysates of S. marcescens CH-1 or 0.5 mM IPTG-inducted E. coli
8
BL21(DE3)(pBG20). pBG20 is a recombinant plasmid containing His-tagged S.
9
marcescens PDH-E1 (AceESm) fusion proteins. The reaction mixture was incubated at
10
4°C for 1 h to allow interaction between GST-PirinSm and the His-tagged fusion
11
proteins. Beads were subsequently washed with PBST buffer, and proteins were
12
eluted with 10 mM glutathione before separation by 12.5% SDS-PAGE and detection
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by immunoblotting.
15
Bacterial two-hybrid screening. The bacterial two-hybrid system used in this
16
study is based on interaction-mediated reconstitution of adenylate cyclase activity in
17
an E. coli host (14). Briefly, interaction between T25-PirinSm and PDH-E1-T18 fusion
18
proteins lead to the cytoplasmic production and assembly of functional adenylate
19
cyclase in E. coli DHM1. This was detected qualitatively by ability of E. coli cells to
20
ferment maltose on maltose-MacConkey agar plates (Difco, Franklin, NJ, USA) to
21
form red colonies (17). IPTG (0.5 mM) was included in the medium to induce full
22
expression and synthesis of hybrid proteins when necessary.
23 24
Protein identification. The in-gel digestion and mass spectrometric analysis
9
1
were performed at EverNew Biotechnology, Inc. (Taipei, Taiwan) using the
2
procedures described previously (30, 34). Briefly, the gel piece was washed in 1 ml of
3
25 mM NH4HCO3 for 10 min and then 1 ml of 25 mM NH4HCO3/50% acetonitrile for
4
10 min. After drying in a SpeedVac (ThermoSavant, Waltham, MA, USA), the gel was
5
incubated with 50 ml of 2% (v/v) β-mercaptoethanol in the dark for 20 min.
6
Following incubation, an equal volume of 10% (v/v) vinyl-pyridine in 25 mM
7
NH4HCO3/50% acetonitrile was added. After 20 min of incubation, the gel was
8
washed several times in 1 ml of 25 mM NH4HCO3 and then dehydrated in 25 mM
9
NH4HCO3 with 50% acetonitrile. The SpeedVac dried gel was then treated with 50 ng
10
of modified trypsin (Promega, Madison, WI, USA) in an adequate volume of 25 mM
11
NH4HCO3 at 37°C for overnight. The resultant peptides were extracted with 200 µl of
12
0.1% formic acid, dried in the SpeedVac and then stored at -20°C until further use.
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Electrospray ionization tandem mass spectrometry was performed using a
14
ThermoFinnigan LCQ Deca ion trap mass spectrometer (Waltham, MA, USA)
15
interfaced with an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA,
16
USA). A 150 × 0.3 mm Agilent ZORBAX 300SB-C18 column (3M particle diameter,
17
300 Å pore size) with mobile phases A (0.1% formic acid in water) and B (0.085%
18
formic acid in acetonitrile) was used. The peptides were eluted at a flow rate of 5
19
µl/min, with gradients that consisted of 5%-16% B in 5 min, 16%-20% B in 40 min,
20
and 20%-65% B in 40 min.
21
The spectra for the eluates were acquired as successive sets of three scan
22
modes, MS, zoom and MS scans, as described previously (30, 34). The MS scan
23
determined the intensity of the ions in the m/z range of 395 to 1605, and a specific ion
24
was selected for the zoom and MS/MS scans. The former examined the charge
10
1
number of the selected ion and the latter acquired the spectrum (CID spectrum or
2
MS/MS spectrum) for the fragment ions derived by collision-induced dissociation
3
(CID). In the first analysis, the most abundant ion in an MS spectrum was selected for
4
the CID experiment; in the second analysis, only the ions with m/z values
5
corresponding to the potential phosphopeptides were selected for the CID experiment.
6
The acquired CID spectra were interpreted using TurboSequest software
7
(ThermoFinnigan) that matched tandem mass spectra against a non-redundant protein
8
database.
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Synthesis and purification of S. marcescens PDH E1, E2 and E3 subunits.
11
PDH subunits E1, E2, and E3 were overproduced in E. coli DH5α cells containing
12
pSC12, pSC19, and pSC20, respectively. Briefly, overnight cultures of E. coli cells
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13
transformed with each recombinant plasmid were diluted 1:10 in fresh LB medium
14
and grown for 2 h at 37°C before addition of 0.1 mM IPTG. After another 3 h of
15
growth, cells were centrifuged and resuspended in lysis buffer (20 mM Tris-HCl, pH
16
8.0; 100 mM NaCl; 1 mM EDTA; 1% TritonX-100). Cells were lysed by sonication
17
followed by centrifugation at 10,000 × g for 20 min at 4°C. The supernatant was then
18
subjected to glutathione sepharose purification (26).
19 20
PDH E1 and PDH enzyme complex activity assays. Determination of PDH
21
E1 was achieved by an assay that measures the rate of reduction of the artificial
22
electron acceptor 2,6-dichlorophenolindophenol (DCPIP) by the E1 component, with
23
pyruvate as a substrate (DCPIP assay) (12). Decrease in absorbance at 600 nm was
24
monitored at 30°C in a suspension comprising 0.2 mM ThDP (thiamin diphosphate), 2
11
1
mM MgCl2, 50 µM DCPIP, 100 mM potassium phosphate (pH 7.0), and spent crude
2
protein extracts (20-50 µg), purified E1 protein or other protein reactants at the
3
concentration of 15 µg each. After incubating at 30°C for 10 min, reactions were
4
initiated by addition of pyruvate (final concentration of 400 µM) followed by
5
monitoring the amount of DCPIP reduced over the reaction period of up to 2 h.
6
DCPIP reduction was calculated by the absorbance change using a molar absorption
7
constant of ε600= 11000 M-1cm-1 for DCPIP (35).
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8
PDH enzyme complex catalytic activity was measured using the PDH assay (5).
9
The PDH assay measured the rate of NADH formation by absorbance at a wavelength
10
of 340 nm at 30°C (5, 10). The reaction was started by addition of final concentrations
11
of 2 mM pyruvate and 0.13 mM CoA into the assay mixture containing 0.2 mM ThDP,
12
1 mM MgCl2, 2.6 mM cysteine HCl, 2.5 mM NAD+, 50 mM potassium phosphate
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(pH 7.0), and 20-50 µg of the reconstituted PDH enzyme complex (E1:E2:E3 molar
14
ratio = 2:2:1) or spent crude cellular extracts. Specific activities were expressed as
15
units (micromoles of NADH formed per min) per milligram of whole cell lysate in the
16
assay.
17 18
Determination of acetyl-CoA concentration. Procedures for determination of
19
acetyl-CoA concentrations followed the protocols of Deutsch et al. (9). Briefly,
20
acetyl-CoA was separated by high pressure liquid chromatography (HPLC) using a
21
C18 column (Mightysil RP-18 GP, Kanto Chemical Co., Tokyo, Japan) and eluted
22
with an interrupted linear gradient of acetonitrile in 0.1 mol/L potassium-phosphate
23
(pH 5.0). Initial conditions were 81% solvent A (0.1 mol/L potassium phosphate, pH
24
5.0) and 19% solvent B (40% acetonitrile in solvent A); a constant flow rate of 0.25
12
1
ml/min was used during separation. The column was equilibrated at 3% of B for 10
2
min between injections. Detection was by absorbance at a wavelength of 254 nm.
3
Retention times of the acetyl-CoA standards were used to identify the peaks on the
4
HPLC chromatograms of each reaction sample. To see the effect of PirinSm on
5
production of acetyl-CoA, the reaction samples, which contained 0.2 mM ThDP, 1
6
mM MgCl2, 2.6 mM cysteine HCl, 2 mM pyruvate, 0.13 mM CoA, 2.5 mM NAD+, 50
7
mM potassium phosphate (pH 7.0), and crude cellular extracts containing 50 µg of
8
protein, were incubated for 1 h at 30°C. Each sample was then applied to HPLC for
9
quantification of acetyl-CoA concentration by peak height with reference to standards
10
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of known acetyl-CoA concentrations.
Cellular ATP concentration assay. The BacTiter-Glo Microbial Assay Kit
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13
(Promega) together with a photon-counting Autolumat luminometer LB953 (Berthold,
14
Dortmund, Germany) for detection of light emission were used for determination of
15
cellular ATP concentration. Briefly, spent cell lysates (100 µl) prepared from fixed
16
amounts of bacteria (V(ml) × OD600nm = 0.8) were mixed thoroughly with 100 µl of
17
substrate solution. This was followed by incubation for 2 min at room temperature.
18
The luminescence was then measured at 5 min intervals, and light emission was
19
recorded for 10 seconds. Results were obtained triplicate as ATP concentrations
20
obtained after calculation against a standard curve at time intervals of 10 min.
21 22
Measurement of NAD+ and NADH concentrations. Dinucleotide extraction
23
was modified from the previously described methods (27). Equal numbers of bacterial
24
cells (determined by volume (ml) × OD600nm unit = 5) were harvested at 5 h
13
1
postinoculation in the late log phase by centrifugation for 15 min at 2,500 × g. After
2
washing the bacterial pellet in PBS, either 1 ml of 0.2 M HCl (NAD+ extraction) or 1
3
ml of 0.2 M KOH (NADH extraction) was added. Samples were boiled for 10 min.
4
After centrifugation (5 min, 10,000 × g, 4°C), cell-free lysates were neutralized.
5
Dinucleotide levels were measured with an enzymatic cycling assay described by
6
Bernofsky and Swan (3). Briefly, 400 µl of cycling buffer (2.2 ml of 623 mM bicin,
7
2.2 ml of 2.6 mM 3-(4,5-dimethyl-thiazoyl-2)-2,5-diphenyltetrazolium bromide
8
[MTT], 2.2 ml of 26 mM EDTA, 1.75 ml of 10.4 mM phenazine ethosulfate [PES],
9
and 0.4 ml ethanol) were added to 400 µl of neutralized extraction solution. After
10
incubation for 5 min at room temperature in the dark, the reaction was initiated by
11
adding 160 µl of 1.3 mg/ml yeast alcohol dehydrogenase (Sigma) and the rate of MTT
12
reduction was monitored spectrophotometrically at a wavelength of 570 nm. The
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intracellular
concentrations
of
dinucleotides
were
determined
by
known
14
concentrations NAD+ and NADH (0, 2.5, 5, 25, and 50 µM). All assays were
15
performed in triplicate from three independent cultures.
16
17
Determination of acetate concentration. The amount of acetate was
18
determined as previously described (31). Briefly, equal numbers of bacterial cells
19
(determined by volume (ml) × OD600nm unit = 5) were centrifuged for 15 min at 2,500
20
× g. The bacterial pellets were suspended in PBS before incubation at 80°C for 15 min
21
to stop the enzymatic reactions. The cell lysates were centrifuged for 5 min at 10,000
22
× g at 4°C and acetate concentrations were determined in the supernatants using a kit
23
purchased from R-Biopharm (Marshall, MI, USA).
14
1
RESULTS
2
A Pirin Ortholog in S. marcescens. During the process of selecting for S.
3
marcescens precocious-swarming mutants by transposon mutagenesis assay (15), a
4
pirin gene homologue in a mutant strain was identified to be interrupted by the
5
transposon (Soo and Lai, unpublished data). The pirin gene in S. marcescens CH-1
6
was subjected to genetic and functional analyses. Through conventional restriction,
7
cloning, and sequencing analyses, a 5-kb DNA fragment containing a putative pirinSm
8
gene was identified in S. marcescens CH-1 (GenBank accession number DQ288954).
9
Genetic maps flanking pirinSm were similar between CH-1 and Db11 (Fig. 1A).
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10
Upstream of the pirinSm gene was an orf predicted to encode a LysM protein domain
11
(2) and downstream of the pirinSm gene was an orf encoding an AraC family protein
12
(29) (Fig. 1A).
C A
13
Extensive computational searches using programs such as NCBI-BLAST and
14
FASTA were performed to identify the S. marcescens Pirin-like proteins from the data
15
banks. S. marcescens Pirin homologues are highly conserved, from many bacterial
16
species to human Pirin (26% identity). Pirin orthologs identified in bacteria include
17
those from Pseudomonas aeruginosa PA14 (54% identity), Acinetobacter sp. (49%
18
identity), Corynebacterium glutamicum (43% identity), Brucella melitensis (37%
19
identity), E. coli (YhhW protein, 37% identity), Ralstonia solanacearum (31%
20
identity), Agrobacterium tumefaciens (27% identity), and Clostridium perfringens
21
(24% identity). The amino acid sequence alignments of S. marcescens CH-1 PirinSm
22
with these Pirin orthologs are shown in figure S1. Based on the results of protein
23
sequence pattern analysis from the MOTIFS (http://motif.genome.jp/) and SMART
24
(http://smart.embl-heidelberg.de/) programs, S. marcescens PirinSm protein displays a
15
1
typical bicupin fold comprising a single N-terminal metal coordination site (pirin
2
domain) and a C-terminal pirin-C conserved domain.
3 4
Interaction of S. marcescens PirinSm with the PDH E1 subunit. Current data
5
gathered can not directly indicate the function of PirinSm in S. marcescens. To
6
characterize PirinSm function, the potential proteins interacting with PirinSm in S.
7
marcescens CH-1 were targeted. A recombinant plasmid pSC10 was constructed in
8
which pirinSm was N-terminally fused with glutathione-S-transferase (GST) to form a
9
GST-PirinSm fusion protein. The pSC10 was subsequently transformed into E. coli
10
DH5α for over-synthesis of the GST-PirinSm protein. After purification, the GST
11
protein pull-down assay was performed. Separation of captured proteins by SDS-
12
PAGE highlighted three major bands with the predicted molecular weights of 99 kDa
D E
T P
E C
C A
13
(a), 76 kDa (b), and 50 KDa (c), after comparison with the negative controls (lane 2,
14
GST-nlpBSm (28) fusion, and lane 3, GST protein only) (Fig. 2).
15
Amino acid sequence analyses using electrospray ionization MS/MS (ESI-
16
MS/MS) were subsequently performed for protein identification. Comparison of the
17
partial amino acid sequences of each protein with non-redundant protein databases in
18
the SEQUEST Browser (http://fields.scripps.edu/sequest/) highlighted three proteins
19
with the highest identity, respectively. They were E. coli pyruvate dehydrogenase
20
(PDH) subunit E1 (band a), PDH subunit E2 (band b), and E. coli 2-oxoglutarate
21
dehydrogenase complex (ODHC) subunit E2 (band c) (Fig. S2). PDH E1 was
22
subsequently selected for further study.
23
The GST pull-down assay was further performed to see whether over-
24
synthesized PDH E1 protein subunit interacted with over-synthesized PirinSm. A
16
1
recombinant plasmid pBG20 encoding a His-Tagged PDH E1Sm (AceESm) fusion
2
protein was constructed. Both pBG20 and pSC10 plasmids were transformed into E.
3
coli BL21(DE3) and E. coli DH5α respectively followed by 0.5 mM IPTG induction
4
for protein over-synthesis. The spent supernatants of whole cell lysates prepared from
5
both cells at a volume ratio of 1:1 were mixed thoroughly and incubated at 4°C for 2 h
6
before addition of glutathione sepharose 4B beads for capturing protein complexes.
7
SDS-PAGE followed by Western blot analysis using anti His-Tag antibody confirmed
8
S. marcescens PDH E1 subunit was pulled down by GST-PirinSm (Fig. 3A).
D E
T P
9
Over-synthesized PDH E1 was subsequently used as the bait to confirm its
10
interaction with PirinSm in S. marcescens CH-1. The recombinant plasmid pSC12
11
encoding the GST-PDH E1Sm fusion protein was transformed to S. marcescens CH-1
12
followed by 0.5 mM IPTG induction and pull-down assay to see whether PirinSm was
E C
C A
13
captured. SDS-PAGE analysis followed by Western blot analysis using mono-specific
14
polyclonal anti-PirinSm antibody confirmed PirinSm as the interactor with PDH E1 (Fig.
15
3B).
16
To confirm the interaction between PirinSm and PDH E1, bacterial two-hybrid
17
assay was further performed. Change of colony color from colorless to pink-red after
18
transforming both pSC17 (pKT25 plasmid (14) containing pirinSm gene) and pSC18
19
(pUT18 plasmid (14) containing PDH E1 aceESm gene) into E. coli DHM1 indicated
20
specific activation of maltose catabolic genes (Fig. 3C), thus confirming positive
21
interaction between these two proteins. In brief, the PDH E1 subunit interacted with S.
22
marcescens PirinSm.
23 24
Inhibition of PDH E1 activity by PirinSm. To see whether the PDH E1 activity
17
1
was affected by over-synthesis of PirinSm, the DCPIP assay (12) was performed. The
2
spent cell lysates of E. coli BL21(DE3) cells containing either pBG20 (PDH E1
3
subunit), pSC10 (PirinSm), or the pGEX vector were prepared when cells were grown
4
to late logarithmic phase at OD600nm = 0.6 under 0.5 mM IPTG induction.
5
Measurements of PDH E1 activities were achieved by mixing both cell lysates (25 µg
6
PDH E1 lysate with 50 µg GST-PirinSm or GST Tag lysate) with externally added
7
pyruvate at the final concentration of 400 µM as the substrate. As shown in figure 4A,
8
the PDH E1 activities of mixed cell lysates from E. coli BL21(DE3)(pSC10) and E.
9
coli
10
BL21(DE3)(pBG20)
30%
T P
lower
than
that
from
E.
coli
E C
BL21(DE3)(pGEX) and E. coli BL21(DE3)(pBG20).
11 12
measured
D E
Inhibition of PDH E1 activity in S. marcescens CH-1 by pirinSm over-
C A
13
expression. As S. marcescens CH-1(pSC15, pirinSm expression under the control of
14
the pBAD promoter) cells were grown to OD600nm = 0.6 in LB broth culture, the
15
amount of PirinSm protein was induced up to 5-fold in the presence of 0.02% arabinose
16
(data not shown), and the PDH E1 activity was reduced up to 40% compared with that
17
from S. marcescens CH-1(pBAD18) (Fig. 4B).
18
We reasoned that the PDH E1 activity might be increased in the S. marcescens
19
CH-1 pirinSm mutant strain. To evaluate this possibility, pirinSm was knocked out by
20
insertion-deletion homologous recombination through an streptomycin (Sm)-resistant
21
Ω cassette (23) in S. marcescens CH-1 to form the mutant strain S. marcescens PC103
22
(Fig. 1B). Southern blot hybridization (data not shown) and Western blot analysis (Fig.
23
1C) confirmed deletion of pirinSm in S. marcescens CH-1. No significant effect on
24
growth dynamics in LB broth cultures was observed between CH-1 and PC103 (data
18
1
not shown). The spent cellular crude extracts of CH-1 and PC103 cells grown to
2
OD600nm = 0.6 were prepared and used in the DCPIP assay. PDH E1 activity in PC103
3
cells was 250% higher, compared to that of CH-1 cells (Fig. 4C). Complementation of
4
S. marcescens PC103 by transforming pSC15 into S. marcescens PC103 cells
5
inhibited the PDH E1 activity (Fig. 4D), while transforming the control vector
6
pBAD18 into S. marcescens PC103 cells did not. Thus, S. marcescens PDH E1
7
activity was inhibited by PirinSm.
D E
T P
8
To characterize the specific inhibition of PDH E1 activity by PirinSm, GST-
9
tagged PDH E1 and GST-tagged PirinSm were purified from E. coli DH5α (pSC12)
10
and E. coli DH5α (pSC10), respectively (Fig. 4E). The DCPIP assay showed that
11
GST-PDH E1 activity measured 30% lower in the presence of GST-PirinSm than that
12
of GST alone or GST-NlpBSm fusion protein (28) (Fig. 4F).
E C
C A
13 14
PirinSm inhibition of PDH enzyme complex activity in S. marcescens.
15
Inhibition of PDH E1 activity by PirinSm strongly suggested that the PDH enzyme
16
complex activity would be inhibited. To confirm this supposition, a total of 50 µg of
17
spent cellular crude extract was prepared from S. marcescens CH-1 and PC103 each
18
and was used in the PDH activity assay. The cellular PDH activity in S. marcescens
19
PC103 cells was 40% higher than that of S. marcescens CH-1 (Fig. 4G). On the other
20
hand, over-synthesis of PirinSm in the S. marcescens PC103 mutant strain lead to
21
inhibition of PDH activity by 30% compared with S. marcescens PC103 containing
22
the control vector (Fig. 4H). To rule out possible background interferences, the in
23
vitro reconstituted PDH enzyme complex comprising E1:E2:E3 at the molecular ratio
24
of 2:2:1 was further used for measurement of PDH activity. As shown in figure 4I, the
19
1
reconstituted PDH activity was inhibited about 35% by GST-PirinSm compared with
2
the GST-NlpBSm and GST alone controls.
3
Increased PDH activity in the pirinSm-deleted mutant strain S. marcescens PC103
4
should result in increased acetyl-CoA production. To evaluate this supposition, spent
5
cellular crude extracts of S. marcescens CH-1 and PC103 were prepared, followed
6
by C18 reverse-phase HPLC separation for determination of acetyl-CoA
7
concentrations. The acetyl-CoA concentration increased about 50% in S. marcescens
8
PC103 in comparison to that of S. marcescens CH-1 (Table 2). Complementation of
9
pirinSm in S. marcescens PC103 by transformation of pSC15 into S. marcescens
10
PC103 cells reduced acetyl-CoA concentration up to 40% compared with that of S.
11
marcescens PC103 (Table 2). Thus, PirinSm inhibited PDH enzyme complex activity
12
in S. marcescens CH-1.
D E
T P
E C
C A
13 14
Cellular ATP concentration and NADH/NAD+ ratio are increased in S.
15
marcescens PC103. Following the process of converting pyruvate into acetyl-CoA by
16
PDH enzyme complex, acetyl-CoA is further oxidized through TCA cycle
17
intermediates, accompanied by synthesis of NADH and FADH2 (4). The majority of
18
the cellular ATP is produced from these reduced NADH and FADH2 compounds after
19
further respiration reactions (25). Alternatively, acetyl-CoA is converted to acetate
20
through dissimilation via the PTA-ACKA pathway and produces less ATP (33). Thus,
21
alteration of the acetyl-CoA concentration should result in a change in cellular ATP
22
concentration. Indeed, cellular ATP concentration of S. marcescens PC103 grown to
23
OD600nm = 0.6 in the late log phase was increased 220% compared with that of S.
24
marcescens CH-1 (Fig. 5A); complementation of S. marcescens PC103 with pSC15
20
1
reduced the ATP concentration to the normal level (Fig. 5B). The increase in ATP
2
concentration in S. marcescens PC103 might have been due to activation of either the
3
TCA cycle or the PTA-ACKA pathway, or both. To clarify this, the ratio of
4
NADH/NAD+, which shows TCA cycle activity and intracellular redox status (31),
5
and cellular acetate concentration were determined. While there was no significant
6
difference in acetate concentration (Fig. 5C), the NADH/NAD+ ratio of S. marcescens
7
PC103 was significantly higher than that of S. marcescens CH-1 (0.759 vs. 0.439, p
