The catabolism of 3,3’-thiodipropionic acid in Variovorax paradoxus strain TBEA6: A proteomic analysis
The catabolism of 3,3'-thiodipropionic acid in Variovorax paradoxus strain TBEA6: A proteomic analysis
Viktoria Heine 0 1
Christina Meinert-BerningID 0 1
Janina L u?ck 0 1
Nadine Mikowsky 0 1
Birgit Voigt 1
Katharina Riedel 1
Alexander Steinbu? chel 0 1
0 Institut f u ?r Molekulare Mikrobiologie und Biotechnologie, Westf a ?lische Wilhelms-Universit a ?t, M u ?nster, Germany, 2 Institut f u ?r Mikrobiologie, Ernst-Moritz-Arndt-Universit a ?t, Greifswald, Germany, 3 Environmental Science Department, King Abdulaziz University , Jeddah , Saudi Arabia
1 Editor: Olaf Kniemeyer, Leibniz-Institut fur Naturstoff-Forschung und Infektionsbiologie eV Hans-Knoll-Institut , GERMANY
Variovorax paradoxus strain TBEA6 is one of the few organisms known to utilize 3,3'-thiodipropionate (TDP) as the only source of carbon and energy. It cleaves TDP to 3-mercaptopropionate (3MP), which is a direct precursor for polythioester synthesis. To establish this process in V. paradoxus TBEA6, it is crucial to unravel its TDP metabolism. Therefore, a proteomic approach with subsequent deletion of interesting genes in the bacterium was chosen. Cells were cultivated with D-gluconate, TDP or 3-sulfinopropionate as the only carbon sources. Proteins with high abundances in gels of cells cultivated with either of the organic sulfur compounds were analyzed further. Thereby, we did not only confirm parts of the already postulated TDP metabolism, but also eight new protein candidates for TDP degradation were detected. Deletions of the corresponding genes (two enoyl-CoA hydratases (Ech-20 and Ech-30), an FK506-binding protein, a putative acetolactate synthase, a carnitinyl-CoA dehydratase, and a putative crotonase family protein) were obtained. Only the deletions of both Ech-20 and Ech-30 led to a TDP negative phenotype. The deletion mutant of VPARA_05510, which encodes the putative crotonase family protein showed reduced growth with TDP. The three genes are located in one cluster with genes proven to be involved in TDP metabolism. Thermal shift assays showed an increased stability of Ech-20 with TDP-CoA but not with TDP. These results indicate that Ech-20 uses TDP-CoA as a substrate instead of TDP. Hence, we postulate a new putative pathway for TDP metabolism. Ech-30 interacts with neither TDP-CoA nor TDP but might interact with other CoA-activated intermediates of the proposed pathway. Further enzyme characterization is necessary to unravel the complete pathway from TDP to 3MP.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: The study was funded by the Deutsche
Forschungsgesellschaft DFG (Grant Number:
STE386/1-12 to AS) and the Open Access
Publication Fund of the University of Muenster. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
The species Variovorax paradoxus belongs to the ?-proteobacteria and occurs in soil and water
as well as on plants [
]. Different strains of this species utilize a variety of metabolic pathways
as its name indicates. Not only are simple carbon sources such as succinate or gluconate
possible nutrients, but xenobiotics and recalcitrant chemicals such as aromatic sulfonates,
polychlorinated biphenyls, chitin or linuron can also be metabolized by different strains of this species.
Furthermore, environmental pollutants such as arsenite, heavy metal ions, and halogenated,
aliphatic or cyclic hydrocarbons are tolerated by some strains. Comparative genomics of
different V. paradoxus strains (B4, EPS, S110 and TBEA6) showed that V. paradoxus strain
TBEA6 possesses four unique gene clusters. These clusters may exclusively enable the strain to
use 3,3?-thiodipropionate (TDP) as the only carbon source [
]. The intermediate resulting
from TDP cleavage, 3-mercaptopropionate (3MP), is a possible precursor substrate for the
biosynthesis of the bioplastic poly(3MP) .
Bioplastic is an important topic in today?s time [
]. Petrol-based plastic is non-degradable
and its production diminishes the stocks of fossil resources. Bioplastics, e.g.
polyhydroxyalkanoates (PHA), are produced from alternative substrates and are microbially degradable. In
polythioesters (PTEs), thioester bonds determine the structure of the biotechnological
produced persistent polymer . Therefore, metabolic engineering of different bacterial strains
was performed to find an optimized genetically modified PTE production strain. To establish
V. paradoxus TBEA6 for the production of PTEs (e.g. to obtain an optimal supply of 3MP),
characterization of the entire TDP metabolism is necessary. Metabolic engineering of strains
as Ralstonia eutropha [
], Advenella mimigardefordensis strain DPN7T , and Escherichia
] was successful and resulted in the production of different homo- and
4, 5, 10, 13
]. While synthesis of poly(3MP) was already established in A.
mimigardefordensis strain DPN7T, production with V. paradoxus TBEA6 has not been accomplished, yet.
Better manageable laboratory conditions concerning V. paradoxus TBEA6 might result in a
more feasible poly(3MP) production.
Parts of the metabolism of TDP, especially the import of TDP and its initial catabolism, are
still unknown. For the import of TDP into the cells, it has been hypothesized that either the
tripartite tricarboxylate transport system (TTT system) or the tripartite ATP-independent
periplasmatic transporter (TRAP system) convey TDP to the cytoplasm [
]. In a previous study,
3MP and traces of 3-hydroxypropionate were identified as expected intermediates of TDP
]. The detailed reaction for TDP cleavage has not been identified until now as
indicated in Fig 1. Initially, a FAD-dependent oxidoreductase Fox (VPARA_05580) was assumed
to catalyze this reaction. Insertions of Tn5::mob in the corresponding gene led to impaired
growth of the mutants with TDP [
]. However, a recent study showed that Fox is not directly
involved in TDP catabolism but has been identified as a 2-hydroxy acid specific dehydrogenase
]. In the downstream pathway 3HP or a similar intermediate enters the central metabolism
while 3MP is processed to 3-sulfinopropionate (3SP) by a cysteine dioxygenase-like
3MP-dioxygenase (Mdo) [
]. The ATP-dependent CoA-ligase SucCD [
] or the
succinyl-CoAdependent CoA-transferase Act  activate the reaction product 3SP by addition of CoA.
This intermediate is cleaved into propionyl-CoA and sulfite by the desulfinase AcdA [
Propionyl-CoA is transferred to the central metabolism while the detoxification of sulfite most
likely occurs at the cell membrane . A Fe/S molybdopterin-dependent sulfite-oxidizing
enzyme (SoeABC) might be responsible for the oxidation of the toxic compound as shown in
Allochromatium vinosum [
] and Variovorax paradoxus B4[
]. Sulfite is oxidized to
sulfate and is most probably exported by a sulfite/sulfate exporter (Pse) [
2 / 22
Fig 1. Synthesis of 3MP from TDP in V. paradoxus TBEA6. The cleavage of TDP has not been characterized until
In late metabolic stages, propionyl-CoA is processed via three different possible pathways:
the malonate semialdehyde pathway, the methylmalonyl-CoA pathway, and the methylcitrate
cycle. The involvement of all three pathways was postulated based on in silico analysis by
Wu?bbeler et al. [
]. In the malonate semialdehyde pathway, several dehydrogenases and hydratases
convert propionyl-CoA via malonate semialdehyde to acetyl-CoA. The second pathway
consists of a semialdehyde dehydrogenase and a methylmalonyl-CoA mutase catalyzing the
reactions via methylmalonyl derivatives to succinyl-CoA. Methylcitrate and methylisocitrate are
intermediates of the third pathway catalyzed by corresponding dehydratases. Succinyl-CoA
and acetyl-CoA from all three pathways then enter the central metabolism.
In this study, we applied several methods such as proteomic analyses, marker-free gene
deletion, growth experiments, and enzyme characterization to elucidate the initial catabolic
reactions with TDP. The resulting protein profiles provided several promising proteins; two of
them were annotated as putative enoyl-CoA hydratases (Ech). Deletion of both Echs resulted
in impaired growth with TDP. Therefore, both genes appeared to be essential for the
utilization of TDP by V. paradoxus strain TBEA6. Their function in TDP metabolism and
degradation was further analyzed during this study. Additionally, a putative crotonase family protein
has been hypothesized to be involved in TDP catabolism, since deletion of this gene reduced
the growth of V. paradoxus TBEA6 with TDP. The study provided new insights in TDP
metabolism, and we are now able to propose a potential reaction pathway from TDP to 3MP and
3 / 22
Relevant geno- and phenotype
Utilizes TDP as sole carbon source
?80lacZ?M15, ?lacX74, deoR, recA1, araD139, galK, aaIU, ?(ara-leu)7697, endA1
thi-1, proA, hsdR17 (rk- mk+), recA1, tra-genes of plasmid RP4 integrated into the genome,
F-, mcrA, ?(mrr-hsdRMS-mcrBC), rpsL, nupG
F?, ompT, hsdSB(rB?, mB?), gal, dcm (DE3)/pLysS (Cmr)
F-, ompT, hsdSB, (rB-,mB-) gal, dcm (DE3), pLysSRARE, Cmr
No growth with TDP
Invitrogen, Darmstadt, Germany
Novagen, Madison, USA
Novagen, Madison, USA
No growth with TDP
No growth with TDP
Reduced growth with TDP
Bla, rep(pMB1), eco47IR
TcR, suicide vector for gene deletion
E. coli expression vector, (N-terminal His-tag, Ampr, T7 promoter)
E. coli expression vector (C-terminal His-tag, Ampr, T7 promoter)
pBR322 ori, f1 ori, His6, Ampr, T7lac, Trx
E. coli expression vector (C-terminal His-tag, Ampr, T7 promoter) expressing ech-30
E. coli expression vector (C-terminal His-tag, Ampr, T7 promoter) expressing ech-20
Novagen, Madison, USA
Novagen, Madison, USA
Materials and methods
Cultivation and cell harvest
E. coli strains were cultivated overnight at 37?C on solid or in liquid Lysogeny Broth
containing ampicillin (75 ?g ml-1) or tetracycline (12.5 ?l ml-1) depending on the resistance of the
strain. V. paradoxus was cultivated overnight at 30?C in mineral salt medium (MSM) [
Nutrient Broth in liquid cultures or for 3 to 5 days on solid medium (solidified with 2%
AgarAgar). MSM was completed with different carbon sources (60 mM gluconate, TDP or 3SP).
Liquid cultures were incubated on a rotary shaker at 130 rpm (New Brunswick Scientific Co.,
Inc., NJ, USA) in Erlenmeyer flasks with baffles to guarantee optimal oxygen supply. Strains
used in this study are listed in Table 1.
To determine the growth phase of liquid cell cultures, the optical density (OD) was
measured at defined time intervals. The OD was determined by three different methods depending
on the experiment. 1) A spectral photometer (Thermo Spectronic GENESYS 20 Visible
Spectrophotometer, Conquer Scientific, San Diego, USA) was used for general OD measurements
(e.g. protein production, proteomic approach). For proteome analysis, liquid cultures were
harvested 6 h after entering the stationary growth phase. 2) A Klett-Summerson photometer
(Manostat Corporation, NY, USA) served for supernatant analyses to reduce the risk of
contamination. 3) For growth experiments, a microplate reader (EPOCH2TC, BioTek
Instruments, Winooski, USA) was used. The conditions resembled the conditions in flasks. The
wells of microtiter plates contained 200 ?l of medium and cells of the different strains. Each
growth experiment was performed in triplicates of each culture and with controls, containing
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no cells. Cells were harvested by centrifugation at 4?C and 7,690 x g for 10 min to 30 min
(Universal 320R, Hettich Lab Technology, Tuttlingen, Germany).
Preparation of protein samples for 2D-PAGE
To obtain the proteins, cell pellets were resuspended in the equivalent volume of Tris/HCl
buffer (50 mM Tris, pH 7.4) containing protease inhibitor and DNase. Afterward, the cells
were disrupted at 4?C and 1000 MPa by five passages through a French press (French Pressure
Cell Press KIN004, Fa. Amicon, Silver Spring, Maryland, USA). The cell debris was removed
by centrifugation (7,690 x g, 4?C, 30 min); the supernatants were retained. Precipitation was
done with acetone containing 20% (wt/vol) trichloroacetic acid and 20 mM DTT for 1 h at
-20?C. After centrifugation, the supernatant was discarded. Pellets were washed with 20 ml
acetone containing 20 mM DTT until the yellow pigment naturally formed by V. paradoxus
TBEA6 was removed from the pellet. The pellets were dried and stored at -20?C.
1st dimension: Isoelectric focusing for 2D-PAGE
Protein pellets were dissolved in 500 to 1000 ?l rehydration buffer A (7 M urea, 2 M thiourea,
4% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 100 mM DTT)
and incubated overnight at room temperature to allow optimal rehydration. The protein
solutions were centrifuged for 5 min at 14,000 x g; the supernatants were retained. The volume
corresponding to 1.5 mg protein was complemented to 200 ?l with rehydration buffer A, and
150 ?l of rehydration buffer B (rehydration buffer A with 0.05% (vol/vol) Bio-Lyte 3/10
Ampholyte (Biorad, Hercules, USA), 0.05% (vol/vol) Triton X-100, and a pinch of
bromophenol blue) were added. Solutions and IPG strips (ReadyStrip IPG Strips, pH 5?8, 17 cm, Biorad,
Hercules, USA) were transferred into a focusing tray and covered with mineral oil. The
following voltages were applied for isoelectric focusing, using a PROTEAN IEF cell (Biorad,
Hercules, USA): 250 V (250 Vh), 500 V (500 Vh), 1,000 V (1,000 Vh), 6,000 V (108,000 Vh), and 500
V (until further use).
2nd dimension: SDS-PAGE for 2D-PAGE
After preparation of the IPG strips, 12.5% (wt/vol) polyacrylamide gels (200 mm x 200 mm x 1
mm) were produced as described before [
]. IPG-strips were equilibrated for 10 min in
SDSequilibration buffer (1.5 M Tris/HCl pH 8.8, 7M urea, 2% SDS, 34.5% glycerol, and a pinch of
bromophenol blue) and then for another 10 min in SDS-equilibration buffer supplemented by
iodoacetamide. The strips were washed in electrode buffer (25 mM Tris/HCl, 192 mM glycine,
0.1% (wt/vol) SDS), which was also used for 2D-PAGE in a DODECA Cell (PROTEAN plus
DODECA Cell, Biorad, Hercules, USA), and fixed onto the gels with sealing solution (1%
agarose (wt/vol) in electrode buffer and a pinch of bromophenol blue). The PAGE was carried out
with 5 V per gel for one hour and afterward with 20 V per gel until the run was finished.
Afterward, gels were stained overnight (0.4% Serva Blue G 250 (wt/vol), 45% methanol (vol/vol),
and 9% acetic acid (vol/vol)) and destained for 10 h (33% (vol/vol) methanol and 10% (vol/
vol) acetic acid). Gels were stored in 10% acetic acid (vol/vol).
Software-based analysis of 2D-gel images
The gels were analyzed via Delta2D software (Decodon GmbH, Greifswald, Germany)
according to the manufacturer?s recommended practices. Four replicates of each condition (different
carbon sources: gluconate, TDP, and 3SP) were produced in two independent biological
experiments. Three gels per condition were selected for further analysis via Delta2D. Gels of
5 / 22
identical conditions were warped to correct differences between the gels. The average gel
images of all three conditions were warped to create a spot mask. Intensities of the spots were
calculated due to their volume percentage in relation to the total level of protein contained in
the gel. After normalization of the spot volumes by the program, statistics were performed to
exclude insignificant spots. Therefore, analysis of variance (ANOVA) was applied with a
pvalue of 0.05. Spots with a standard deviation higher than 30% were excluded from the
analysis. Spots of the different conditions were compared and set into relation. The values displayed
the differences in the abundance of certain spots regarding the certain conditions (carbon
sources) in contrast. Spots, which exhibited a ratio higher than two in gels of cells cultivated
with TDP or 3SP, were filtered and analyzed via matrix laser
desorption/ionization-time-offlight-tandem mass spectrometry (MALDI-TOF-MS/MS).
Selected spots were cut from the SDS-gels and stored in 10% (vol/vol) acetic acid. The samples
were prepared for MALDI-TOF-MS/MS analysis as described previously [
]. The analysis
was performed with a 4800 Proteomics Analyzer (AB Sciex, Framingham, MA, USA). Spectra
were recorded in the reflector mode with a focus mass of 2000 Da (mass range: 900 Da to 3700
Da). Calibration of mass spectrometry data was performed as already described [
proteome database of V. paradoxus strain TBEA6 was used for the identification of peaks with
peak lists from MS and MS/MS, using the Mascot engine (version 18.104.22.168).
Isolation and transfer of DNA
Depending on the experiment, DNA was isolated with different kits following the
manufacturer?s instructions. Cells were harvested from liquid cultures or agar plates and treated as
stipulated. Genomic DNA was isolated using the NucleoSpin Tissue Kit (Machery-Nagel GmbH
Co. KG, Du?ren, Germany), plasmid DNA was extracted with the GeneJET Plasmid Miniprep
Kit (Fermentas, St. Leon-Tor, Germany). DNA fragments were purified from reaction
mixtures by peqGOLD gel extraction kit (PEQlab Biotechnology GmbH, Erlangen, Germany). To
transfer DNA to E. coli strains, competent cells were prepared and transformed using the
CaCl2 method [
]. Conjugation was performed [
] to transfer plasmids from E. coli to V.
Modification of DNA
DNA fragments were amplified via PCR in a thermocycler (peqSTAR 2x Gradient
Thermocycler, Peqlab Biotechnologie GmbH, Erlangen, Germany) using taq (Biomix, Bioline, London,
UK) or Phusion High Fidelity polymerase (Fermentas, St. Leon-Roth, Germany). Used
oligonucleotides were produced by MWG-Biotech (Ebersberg, Germany) and are listed in S1 Table.
Restriction endonucleases (Fermentas, St. Leon-Rot, Germany) were used for DNA digestion;
T4-Ligase (Invitrogen, Karlsruhe, Germany) was used for ligation.
DNA samples were sequenced by MWG-Biotech (Ebersberg, Germany). Characterization of
the sequences was performed using the Seqman software (DNASTAR, Wisconsin, USA).
Construction of gene deletion suicide plasmids
Up- and downstream flanking regions of the target genes were amplified using the
oligonucleotides listed in S1 Table. Both flanks were ligated and amplified again with the corresponding
6 / 22
primers. The constructs were integrated into the suicide plasmid pJQ200mp18Tc [
pJET1.2/blunt as a subcloning vector. Plasmids were multiplied in E. coli TOP10 and
transferred to E. coli S17-1 for further use.
Gene deletion using the sacB system
The suicide plasmid was transferred from E. coli S17-1 to V. paradoxus TBEA6 via spot mating
] for heterologous recombination. Relevant deletion mutants were identified by inoculation
on solid nutrient broth medium containing 15% (wt/vol) sucrose (growth), MSM containing
Tc and gluconate (no growth), and MSM containing TDP (no growth). Deletion of the target
gene was verified by PCR and sequencing. Correct mutants showed no PCR product with
internal primers and a truncated PCR product with external primers (S2 Table). Internal
primers bind inside the gene, external primers bind outside of the flanking regions.
The consumption of TDP was analyzed in cultures of V. paradoxus TBEA6 and Ech mutant
strains. First, cell mass was generated via cultivation with MSM containing 60 mM succinate
until the cultures reached the late exponential growth phase. Cells were harvested and
transferred to MSM with 50 mM TDP and 10 mM succinate. After medium exchange, samples of 5
ml were withdrawn at specific time intervals (t = 0, 24, 48, 72, 144 h) and centrifuged. Control
flasks without carbon source or without cells were applied to exclude contamination and
detect spontaneous degradation of TDP to 3MP in the medium. The supernatants were
analyzed by gas chromatography (GC; Series 6890 GC-System, Hewlett Packard GmbH,
Dortmund, Germany) using a SGE BP21 capillary column with a split ratio of 1:20, a pressure of
86.5 kPa and a split temperature of 250?C (SGE, Darmstadt, Germany). Two ?l of the sample
were injected at a hydrogen flow of 18.6 ml/min. For sample preparation, 2 ml of the
supernatant were lyophilized, solved (2 ml methanol-sulfuric acid (20:3, (vol/vol)) and 2 ml
chloroform), and incubated for 3 h at 100?C. Afterwards, 2 ml of water were added and vortexed.
The organic phase was extracted and measured.
Heterologous expression of pctRe, ech-20 and ech-30
For heterologous expression of pctRe, E. coli BL21 (DE3) pLysS was transformed with
pET19b::pctRe. The cells were cultivated in a preculture overnight in 20 ml of LB medium
containing 75 ?g/ml ampicillin. The main culture was inoculated with 1% (vol/vol) of the pre-culture
and incubated at 30?C. Expression of pctRe was started by the addition of 1 mM IPTG at an
OD600 0.4 as described previously by Lindenkamp et al. [
]. Purification of recombinant
PctRe was performed as described in detail [
] using Ni-Sepharose columns (His SpinTrap;
GE Healthcare, Munich, Germany).
Heterologous production of Ech-20 and Ech-30 was accomplished by transformation of E.
coli BL21 (DE3) pLysS with pET-23a(+)::ech-30 and pET-32a(+)::ech-20. The freshly
transformed cells were incubated in 20 ml LB medium overnight. From this preculture, 1% (vol/
vol) was used to inoculate the main culture of 50 ml or 100 ml autoinduction medium (ZYP,
]). The cultures were incubated for approximately 18 h on a rotary shaker at 130 rpm.
Subsequently, cells were harvested (7,360 x g, 15 min, 4?C) and disrupted by three to five passages
through a FrenchPress (4?C, 1000 MPa). Cell debris was removed by centrifugation, and the
recombinant Echs were purified using Ni-Sepharose columns (His SpinTrap; GE Healthcare,
Munich, Germany). Following the manufacturer?s instructions we used sodium phosphate
(NaP) buffer (binding buffer: 20 mM NaP, 20 mM imidazole, 500 mM NaCl; wash buffer: 20
mM NaP, 100 mM imidazole, 500 mM NaCl; elution buffer: 20 mM NaP, 500 mM imidazole,
7 / 22
500 mM NaCl). For protein storage and further experiments, the elution buffer was exchanged
for 20 mM NaP buffer (pH 7.4) containing 100 mM NaCl using Vivaspin 500 columns
(Sartorius AG, Go?ttingen, Germany).
Synthesis of CoA-esters
The transfer of CoA from acetyl-CoA to TDP was accomplished using recombinant PctRe as
described by Volodina et al. [
]. Accordingly, 1 mM acetyl-CoA, 2 mM TDP, and 20 ?g of
PctRe were mixed in 100 mM Tris/HCl buffer (pH 8,0); the final volume amounted to 1 ml.
Samples were incubated for 45 min at 30?C. To stop the reaction and denature the protein,
180 ?l of trichloroacetic acid were added. The denatured PctRe was removed by centrifugation,
and the formed TDP-CoA was purified as described in detail by Eggers et al. [
]. First, the
used Sep Pak C18 Classic column (Waters Corporation, Milford, USA) was rinsed two times
with 2.5 ml of 80% (vol/vol) methanol. The columns were equilibrated twice with 2.5 ml of a
50 mM ammonium acetate solution (pH 7.4). Additionally, the ammonium acetate solution
was added to the samples in a ratio of 1:10 and applied to the columns. Subsequently, the
columns were washed twice with 2.5 ml of a mixture of 80% methanol and 20% ammonium
acetate buffer (50 mM, pH 4.7). The CoA esters were finally eluted with 2.5 ml of 80% (vol/vol)
methanol and dried in a vacuum furnace at 30?C.
High performance liquid chromatography/ mass spectrometry (HPLC/MS)
HPLC/MS was used to analyze the synthesized TDP-CoA. Therefore, the samples were
acidified by addition of 0.1% formate and analyzed in an UltiMate 3000 HPLC apparatus (Dionex
GmbH, Idstein, Germany) directly connected to an LXQ Finnigan mass spectrometer
(ThermoScientific, Dreieich, Germany). An Acclaim 120 C18 Reversed-Phase LC Column (4.6 x
205 mm, 5 ?m, 120 ? pores; Dionex GmbH, Idstein, Germany) was used at 30?C. Ammonium
acetate buffer (50 mM, pH 4.7; eluent A) and 100% methanol (eluent B) served as eluents. The
flow rate was set to 0.5 ml/min. Ramping was performed as follows: equilibration was started
with 80% eluent A and 20% eluent B. After injection, within a period of 20 min, eluent B was
increased in a gradient to a final concentration of 85%. Within the next 10 min, the
concentration of eluent B was reduced to 20% and held for a duration of another 10 min. Detection of
CoA esters was done at 259 nm with a photodiode array detector. The device was tuned by
direct infusion of a 1 mM CoA trilithium salt solution at a flow rate of 10 ?l/min into the ion
trap of the mass spectrometer to optimize the ESI-MS system for generation of protonated
molecular ions of CoA derivatives. The tuning parameters were set as follows: capillary
temperature, 300? C; sheat gas flow, 12 liters/h; auxiliary gas flow, 6 liters/h; and sweep gas flow, 1
liter/h. The mass range was set to m/z 50 to 1,000 Da when running in the scan mode. The
collision energy in the MS mode was set to 30 V.
Thermal shift assay
Thermal shift assays were used to elucidate a stabilizing effect of putative ligands on Ech-20
and Ech-30. Therefore, 2 ?M of protein, 2 mM of the potential ligand, and 5 x SyproOrange
(5000 x stock solution, Sigma-Aldrich, MO, USA) were mixed in a final volume of 20 ?l using
a Tris/HCl (100 mM, pH 8.0) buffer system. Samples were put into 48 well plates
(MicroAmpOptical 48-Well reaction plate, Applied Biosystems, Foster City, CA, USA) and sealed with
optical adhesive films (MicroAmp 48-well Optical Adhesive Film, PCR Compatible, Applied
Biosystems, Foster City, CA, USA). A StepOne real-time PCR system (Applied Biosystems,
Forster City, CA, USA) was used to record the fluorescence during the temperature range of
20?C to 90?C following the protocol from Vivoli et al. [
8 / 22
Results and discussion
The aim of this study was the characterization of the degradation of TDP to 3MP and 3HP. A
proteomic approach was performed to detect proteins, influenced by growth of V. paradoxus
TBEA6 using TDP as the only carbon source. Deletion of genes coding for two Echs and a
crotonase family protein led to significant phenotypes, which were confirmed by further
experiments. Results from the different approaches are described in this chapter to provide thorough
Proteome analysis of V. paradoxus TBEA6
V. paradoxus TBEA6 cells were cultivated in MSM with gluconate, TDP or 3SP (60 mM) as
sole carbon source, respectively, to display differences in protein profiles. Comparisons were
drawn between the 2D gels of cells cultivated with TDP and gluconate, 3SP and gluconate, and
TDP and 3SP (Fig 2). Cells of all three cultures were harvested 6 h after reaching the stationary
phase. The experiment was repeated to confirm the results of the first biological experiment
(S1?S3 Figs; S3 and S4 Tables).
Identification of proteins with increased abundance during cell growth
with TDP and 3SP in comparison to gluconate
To characterize the initial reactions of TDP catabolism in detail, 2D gels of cells cultivated with
TDP or 3SP were compared to gels of cells cultivated with gluconate. Furthermore, the
comparison of the protein profiles of TDP and 3SP (Fig 2) might provide specific evidence to the
process itself, because the detected proteins can be related immediately to TDP catabolism.
The other comparisons relate to the complete catabolism of TDP, also including downstream
steps of the postulated catabolism.
To identify proteins potentially involved in the mechanism, the protein spots were analyzed
via Delta2D Software. Quantitation tables were generated, displaying the normalized spot
volumes (%V) and the ratios between the spots of different conditions (S3 and S4 Tables; S1?S3
Figs). A spot mask filtered spots with ratios higher than 2 or lower than 0.5 and highlighted the
most promising proteins. For our purpose, only spots with a ratio higher than 2 were
considered. An increased expression of genes with TDP as a carbon source was expected to indicate
genes connected to TDP catabolism. From 173 detected spots, 98 fulfilled the requirements.
For 79 of these selected spots, proteins were identified and assigned to locus tags in the genome
of V. paradoxus TBEA6 (Table 2).
The better part of the detected proteins (59%) act in metabolic pathways, while the others
catalyze processes in the biosynthesis of building blocks (28%), transport (7%), transcription
(2%), DNA modification (2%), cellular stress response (1%), and protein folding (1%). The
detected proteins were analyzed via in silico analysis and examined regarding their potential
function in TDP metabolism.
Important reactions of the TDP catabolism from 3MP and 3SP to propionyl-CoA have been
described in detail [
]. The proteome analysis of this study displayed the pathway very clearly.
Amongst all detected proteins, the 3-mercaptopropionate dioxygenase (Mdo) [
acylCoA dehydrogenase-like desulfinase (AcdA) [
], and the succinyl-CoA-dependent CoA
transferase (Act)  occurred with a high abundance in the 2D gels (Table 3). Regarding the
metabolic part leading from propionyl-CoA to the central metabolism, we postulated three
putative pathways from in silico analysis [
]. With the proteomic approach, we detected at least
9 / 22
Fig 2. 2D-gel dual views and relevant carbon sources. Left: Dual views were modeled by overlay of 2D-gels carrying
the proteomic composition during cell growth with one specific carbon source. Proteins (1.5 mg per gel) were
separated by isoelectric point between pH 5 and pH 8 (first dimension) and molecular weight (second dimension).
Gels were stained with Coomassie Brilliant Blue and analyzed via Delta2D Software. Interesting spots were isolated and
examined via MALDI-TOF. These spots are highlighted in this figure. A) TDP (orange) vs gluconate (blue); B) 3SP
(orange) vs gluconate (blue); C) TDP (blue) vs 3SP (orange). Right: Gluconate, TDP, 3SP or succinate were used for
several growth experiments to identify the impact of the organic sulfur compounds on the proteome.
one enzyme of each postulated pathway (Table 3). For the malonate semialdehyde pathway,
participation of Ech-30 and the semialdehyde dehydrogenase were observed. The
semialdehyde dehydrogenase putatively performs two steps in the methylmalonyl-CoA pathway as
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TDP vs 3SP
well. The 2-methylcitrate synthase, the 2-methylcitrate dehydratase, and the methylisocitrate
lyase are possibly involved in the the methylcitrate cycle. The appearance of all these enzymes
supports the three postulated pathways. It is likely that they occur simultaneously. The
resulting intermediates, succinyl-CoA and propionyl-CoA are common metabolic intermediates.
Therefore, the three described pathways might as well be active during other metabolic
reactions with alternative initial substrates.
Proteins selected for gene deletion
Nine different proteins aroused attention due to their putative function and their high
abundance in 2D gels of cells grown with TDP or 3SP in comparison to gluconate as the only
carbon source (Table 4).
A putative crotonase family protein and two putative enoyl-CoA hydratases (Ech-20 and
Ech-30) showed high abundance in 2D gels from cultures with both organic sulfur compounds
in contrast to the control (gluconate). Also a carnitinyl-CoA dehydratase, a putative
acetolactate synthase IlvX, and two putative FAD-linked oxidoreductases were analyzed due to their
overrepresentation in the proteome of cells grown with TDP and 3SP. An FK506-binding
protein FKBP and a putative metallo hydrolase exhibited an increased spot volume in TDP gels.
Deletion was accomplished for each corresponding gene, except for VPARA_27760. It is likely
that this enzyme is essential for the survival of the cell. The gene encoding the putative
crotonase family protein (VPARA_05510) was deleted due to its location in the cluster comprising
genes responsible for TDP catabolism (Fig 3).
Ratio Vol% TDP / Vol% Ratio Vol% 3SP / Vol% Ratio Vol% TDP / Vol
Gluc Gluc % 3SP
26.11 8.02 3.25
5.58 3.39 3.63
Putative acetolactate synthase large subunit ilvX
Putative FAD-linked oxidoreductase
FK506-binding protein (22.214.171.124) fbp
Putative FAD-linked oxidoreductase
Putative metallo-hydrolase (3.-.-.-)
Ratio Vol% TDP /
Ratio Vol% 3SP /
Ratio Vol% TDP /
Only the deletion of ech-20, ech-30, and the gene encoding the crotonase family protein
resulted in an altered phenotype with TDP as the only carbon source. Deletion mutants lacking
VPARA_05540, VPARA_05550, VPARA_21730, VPARA_24900, or VPARA_27740 showed
no change in growth with TDP (not shown). Still, they might be involved in the general sulfur
metabolism (e. g. amino acid biosynthesis, detoxification) due the high abundance of the
respective proteins. Some of these proteins were detected in the same spots as proteins crucial
for TDP metabolism. High abundance of the carnitinyl-CoA dehydratase (VPARA_05540)
and the acetlocatate synthase (VPARA_05550) might be related to their genetic vicinity (Fig
3) to important genes of the TDP metabolism. For the other genes (VPARA_21730,
VPARA_27740, VPARA_27760), no connections to the TDP catabolism were identified.
Enoyl-CoA hydratases (VPARA_05520, VPARA_05530) and a crotonase
family protein (VPARA_05510)
Ech-20 and Ech-30 were of special interest, as they seemed to be involved in TDP metabolism.
Deletion mutants lacking one or both ech genes were unable to grow with TDP. Growth with
3SP and gluconate was unaffected. The observed phenotype was confirmed in liquid cultures
Fig 3. Gene cluster relevant for TDP degradation as indicated by proteome analysis, comprising mdo, act and
acdA. Proteins with high abundance during cultivation with TDP identified by proteome analysis are marked in
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Fig 4. Growth curves of the wildtype V. paradoxus TBEA6 wildtype in comparison to the mutants ?ech-20 and ?ech-30. Cells were cultivated in MSM
containing 60 mM gluconate, TDP or 3SP. V. paradoxus TBEA6 with gluconate (dark green), V. paradoxus TBEA6 with TDP (orange), V. paradoxus TBEA6
with 3SP (dark blue) (in A and B), A) V. paradoxus TBEA6 ?ech-20 with gluconate (light green), V. paradoxus TBEA6 ?ech-20 with TDP (yellow), V.
paradoxus TBEA6 ?ech-20 with 3SP (light blue). B) V. paradoxus TBEA6 ?ech-30 with gluconate (light green), V. paradoxus TBEA6 ?ech-30 with TDP (red),
V. paradoxus TBEA6 ?ech-30 with 3SP (light blue). The experiment was performed in triplicate. Standard deviations are indicated as error bars.
In an experiment analyzing the metabolism of TDP by GC analysis of the substrate (Fig 5),
the ech deletion mutants were cultivated in comparison to the wildtype. Both mutant strains
and the wildtype were cultivated with succinate to a certain optical density. Then, cells were
Fig 5. Supernatant analysis of the wildtype V. paradoxus TBEA6 in comparison to the mutants ?ech-20 and
?ech30. Cell mass was generated with MSM containing 60 mM succinate for 26 h (not displayed in the graph). Medium
was exchanged after reaching 400 Klett Unit [KU]; subsequently, cells were grown with 10 mM succinate and 50 mM
TDP. Samples were withdrawn at t = 0, 24, 48, 72, 144 h and analyzed via GC. V. paradoxus TBEA6 (blue), V.
paradoxus TBEA6 ?ech-30 (green), V. paradoxus TBEA6 ?ech-20 (orange). Top: growth curve after medium exchange;
bottom left: TDP-concentration in the supernatant detected via GC; bottom right: 3MP-concentration in the
supernatant detected via GC.
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Fig 6. Growth of V. paradoxus TBEA6 and V. paradoxus ?05510. Growth was monitored at 600 nm. Cells were
cultivated in MSM containing 60 mM succinate (TBEA6 (dark green); ?05510 (light green)) or 3,3?-thiodipropionate
(TDP, TBEA6 (dark orange); ?05510 (light orange)) as the only carbon source. The experiment was done in triplicate;
standard deviations are indicated as error bars.
transferred to TDP-containing medium. Samples were withdrawn at different time points to
analyze the consumption of TDP by the strains.
The growth curve after medium exchange showed how the wildtype cultures outstretched
the exponential growth phase while both deletion mutants entered the stationary phase at 24 h
to 48 h after medium exchange. At this time point, succinate (10 mM) was metabolized
completely. The GC measurements exhibited a small amount of 3MP (4 mM) from the
beginning of the experiment. Probably, TDP disintegrates in small amounts to 3MP due to
cultivation conditions (temperature, oxygen). The wildtype immediately utilizes TDP and 3MP. The
supernatant of both mutant cultures showed a decrease neither of TDP (45 mM) nor of 3MP
(4 mM) until the end of the experiment as recorded for the cell-free controls. It can be
concluded that TDP was not consumed by the ech deletion mutants. Consequently, Ech-20 and
Ech-30 are involved in TDP degradation. Their genetic location (Fig 3) supports this
hypothesis. The cluster comprises genes crucial for the metabolism of 3SP in TDP degradation: mdo,
act, and acdA [
1, 2, 17, 19
The deletion of another gene of this cluster, coding for a putative crotonase family protein
(VPARA_05510), caused a reduced ability to consume TDP as observed in further cultivation
experiments (Fig 6). Weak growth of the mutant with TDP as the exclusive carbon source
indicated that this enzyme is also involved in the degradation of TDP.
Based on these results, a preliminary pathway including Ech-20 and Ech-30 and the
crotonase family protein was hypothesized. Participation of other enzymes and various possible
reaction intermediates complicate the design of putative pathways (Fig 7) based on other
]. It is most likely that at least one acyl-CoA dehydrogenase plays a role during
the process, since enoyl-CoA hydratases usually act on trans-enoyl-esters . Some
corresponding homologous genes are located in the gene cluster comprising important genes
responsible for TDP degradation (Fig 3) (VPARA_05480 and VPARA_05500). Furthermore,
acyl-CoA synthetases, hydroxyacyl-CoA dehydrogenases or ketoadipyl-CoA thiolases are
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Fig 7. Hypothesis for the degradation of TDP. Possibly, TDP is converted to TDP-CoA by a putative formyl-CoA
transferase. Presumably, a double bond is introduced to the molecule after the elimination of two protons by a putative
acyl-CoA dehydrogenase. Afterward, two variants of the catabolism were postulated based on the protein profile from
the proteome analysis. Variant I.: Degradation of TDP via formation of TDP-CoA and subsequent ?-elimination
resulting in the formation of acetyl-CoA by the enoyl-CoA hydratase homologs, the crotonase family protein homolog,
a putative 3-hydroxypropionic acid dehydrogenase, and a semialdehyde dehydrogenase homolog. Variant II.:
Degradation of TDP via ?-oxidation resulting in the formation of formyl-CoA and 3-mercaptopropionate by the
enoyl-CoA hydratases, a putative carnitinyl-CoA dehydratase, ?-keto-thiolase homologs, and a putative crotonase
family protein. The already described catabolism of 3-mercaptopropionate is shown in the grey box.
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Fig 8. Synthesis of TDP-CoA and application in a thermal shift assay with Ech-20. A) Analysis of TDP-CoA via HPLC/MS using an AcclaimTM 120
C18 column. Detection was done at 259 nm with a photodiode array detector. TDP-CoA was detected at 12.52 min (upper diagram) and analyzed via mass
spectrometry with 928.24 m/z (lower diagram). B) Heterologous production of Ech-20 in E. coli Bl21 (DE3) pLysS pET-32a(+)::ech-20. Purification of
Ech20 was performed via IMAC. 40 ?g of protein were loaded onto the gel, except for elution samples of which only 5 ?g were applied to the gel. C) Shift in the
melting temperature of Ech-20 (2 ?M) in presence of 2 mM TDP-CoA (red) and 2 mM TDP (green) shown in a thermal shift assay. The assay was
performed with 2 ?M protein in presence of 2 mM TDP-CoA in 100 mM Tris/HCl (pH 8). Control without protein (lines without peaks; green (TDP) and
red (TDP-CoA)), control without ligand (blue).
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Each pathway starts with the activation of TDP with CoA. Therefore, TDP-CoA was
synthesized enzymatically employing the propionyl-CoA transferase of Ralstonia eutropha (PctRe)
(Fig 8). The purified TDP-CoA was subsequently used in thermal shift assays with Ech-20 and
Ech-30 to elucidate a putative interaction of the enzymes and the CoA-ester. Thereby, a shift
in the melting temperature of Ech-20 in the presence of TDP-CoA was detected (Fig 8)
whereas no shift was observed in presence of TDP (Fig 8). This supports the assumption that
activation of TDP with CoA is necessary for utilization by Ech-20. In vitro experiments are
necessary to verify the enzyme?s impact in TDP-CoA hydrolysis. For Ech-30, no shift was
detected with TDP-CoA or TDP (see supporting information, S4 Fig, S5 Fig). It is possible that
this enzyme catalyzes a different step in TDP consumption. We also investigate the possibility
that both enoyl-CoA hydratases might form a functional unit exhibiting a heterodimeric or
heterotetrameric structure, as described by Yang et al. for a distinct MaoC-like enoyl-CoA
hydratase from Mycobacterium tuberculosis [
]. This could be a reason why we can observe
the interaction of TDP-CoA with Ech-20 but could not show any activity of the enzyme in a
first attempt of an in vitro enzyme assay.
The presented findings confirm TDP-CoA as an intermediate of the TDP catabolism and
delivered new insights into possible mechanisms of the TDP catabolism. Starting from
TDP-CoA or its deprotonated form, one of the metabolic pathways of TDP could be the
?elimination-like pathway [
] (Fig 7, variant I.). Ech-20 could mediate the first step and
use TDP-CoA as a substrate. Regarding the missing interaction of Ech-30 with TDP-CoA, a
reaction with another intermediate of the pathway is likely. Acrylyl-CoA would be the
compound of choice, relating to variant I. of our postulated pathway. It is oxidized to 3HP-CoA
which could be hydrolyzed to 3HP by the crotonase family protein. Variant II. would be
similar to a ?-oxidation-like pathway [
38, 42, 45
] (Fig 7, variant II.). TDP resembles the
dicarboxylic acid pimelic acid , a substrate degraded via ?-oxidation. Therefore, it is possible that
TDP is catabolized via a similar pathway. Also on this route, one of the Ech (probably Ech-20)
and the crotonase family protein could participate. The degradation products of this pathway
would be 3MP and formyl-CoA. Different from the other variant, 3HP would not be an
intermediate of TDP degradation. Further studies are necessary to reinforce our hypotheses,
unravel the function of the remaining enzymes (e.g. hydroxybutyryl-CoA hydrolase), and
determine the actual pathway.
This study showed that TDP consumption affects several different cellular processes.
Therefore, many of the corresponding proteins showed a high abundance during proteome analysis.
Genes encoding high abundant proteins identified by proteome analysis were deleted.
Thereby, three mutants lacking a crotonase family protein (VPARA_05510) or one of two
Echs (VPARA_05520 and VPARA_05530) exhibited impaired growth using TDP as the only
source of carbon. Further investigations such as growth experiments and supernatant analysis
of the mutants as well as the stabilization of Ech-20 with TDP-CoA confirmed the results from
gene deletion. We are now able to postulate the significance of Ech-20 of V. paradoxus TBEA6
for conversion of TDP to 3MP. Also for Ech-30 and the crotonase family protein, we were able
to assign a potential function. Comparing the new pathway with the formerly postulated one,
we gained new insights into the catabolism of TDP, its modification, and further degradation.
S1 Table. Primers used for generation of suicide plasmids for marker-free gene deletion.
The designation contains the locus tag, downstream (Do) or upstream (Up) location of the
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respective flanking sequence, and forward (FOR) or reverse (REV) orientation of the primer.
Furthermore, melting temperatures (TM) are included in the table. Restriction site are inserted
into the primer in front of the sequence for cloning (underlined).
S2 Table. Primers used for verification of marker-free gene deletion. The designation
contains the locus tag, binding of the primer inside (In) or outside (Ex) of the deleted gene, and
forward (FOR) or reverse (REV) orientation of the primer. Furthermore, melting temperatures
(TM) are included in the table.
S3 Table. Quantitation table of the first biological experiment. Displayed are the
normalized and mean normalized spot volumes of spots on gels from cultivations with gluconate in
contrast to TDP and 3SP, ratios of mean normalized volumes, spot labels, and detected
proteins (MALDI-TOF-MS/MS) within respective spots (Accession number). Some accession
numbers are missing for spots that were not identified via MALDI-TOF-MS/MS.
S4 Table. Quantitation table of the second biological experiment. Displayed are the
normalized and mean normalized spot volumes of spots on gels from cultivations with gluconate in
contrast to TDP and 3SP, ratios of mean normalized volumes, and detected proteins
(MALDI-TOF-MS/MS) within respective spots (Accession number). Some accession numbers are
missing for spots that were not identified via MALDI-TOF-MS/MS. Identified spots from the
already described TDP metabolism are highlighted in blue; protein spots that belong to genes
deleted during this study are highlighted in orange; proteins from both, which were found in
the same spot, are marked in grey.
S1 Fig. 2D-gels from the second biological experiment with spot labels. Cells cultivated with
Gluconate and TDP in comparison were disrupted and proteins extracted for 2D-gel analysis.
Proteins (1.5 ?g per gel) were firstly separated by isoelectric point (pH 5 to pH 8) and secondly
by molecular weight. Proteins were stained with Coomassie Brilliant Blue, scanned, labeled,
and analyzed via the Delta2D Software. Spot labels are displayed on the fused images.
S2 Fig. 2D-gels from the second biological experiment with spot labels. Cells cultivated with
Gluconate and 3SP in comparison were disrupted and proteins extracted for 2D-gel analysis.
Proteins (1.5 ?g per gel) were firstly separated by isoelectric point (pH 5 to pH 8) and secondly
by molecular weight. Proteins were stained with Coomassie Brilliant Blue, scanned, labeled,
and analyzed via the Delta2D Software. Spot labels are displayed on the fused images.
S3 Fig. 2D-gels from the second biological experiment with spot labels. Cells cultivated with
TDP and 3SP in comparison were disrupted and proteins extracted for 2D-gel analysis.
Proteins (1.5 ?g per gel) were firstly separated by isoelectric point (pH 5 to pH 8) and secondly by
molecular weight. Proteins were stained with Coomassie Brilliant Blue, scanned, labeled, and
analyzed via the Delta2D Software. Spot labels are displayed on the fused images.
S4 Fig. Purification of Ech-30. Heterologous expression was performed using cells of E. coli
BL21 (DE3) pLysS which were transformed with pET23a::ech-30. ZYP autoinduction medium
was inoculated with freshly transformed cells and the cultures were incubated overnight at
18 / 22
30?C on a rotary shaker at 130 rpm. Purification was achieved using His Spin Trap columns
(GE Healthcare) using 100 mM Tris/HCl buffer (pH 8.0 with 500 mM NaCl and different
concentrations of imidazole. Cells were mixed with binding buffer (20 mM imidazole) before cell
disruption and afterwards cell debris was removed by centrifugation (cell-free lysate). The
cellfree lysate was loaded onto the columns (flow through) and washed with the above mentioned
Tris/HCl Buffer with the following concentrations of imidazole: wash fraction I, 50 mM
imidazole; wash fraction II, 100 mM imidazole; wash fraction III and IV, 200 mM imidazole. The
protein was eluted using the Tris/HCl buffer with 500 mM imidazole.
HCl (pH 8).
S5 Fig. Thermal shift assay of purified Ech-30 using TDP (green) and TDP-CoA (red) as
ligands. The blue curve represents the no ligand control. The lines without the peaks represent
the no protein controls, also with addition of TDP (green) and TDP-CoA (red). The assay was
performed in triplicate with 2 ?M protein in presence of 2 mM TDP-CoA in 100 mM Tris/
Conceptualization: Viktoria Heine, Christina Meinert-Berning, Alexander Steinbu?chel.
Data curation: Viktoria Heine, Christina Meinert-Berning, Janina Lu?ck, Birgit Voigt.
Formal analysis: Viktoria Heine, Christina Meinert-Berning.
Investigation: Viktoria Heine, Christina Meinert-Berning, Janina Lu?ck, Nadine Mikowsky.
Methodology: Viktoria Heine, Christina Meinert-Berning, Janina Lu?ck, Birgit Voigt.
Supervision: Christina Meinert-Berning, Alexander Steinbu?chel.
Writing ? original draft: Viktoria Heine.
Writing ? review & editing: Christina Meinert-Berning, Katharina Riedel, Alexander
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