Proteome Analysis of Sulfolobus solfataricus P2 Propanol Metabolism Poh Kuan Chong,†,‡ Adam M. Burja,†,§ Helia Radianingtyas,†,§ Alireza Fazeli,| and Phillip C. Wright*,† Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK, and Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield, S10 2SF, UK Received October 31, 2006
Sulfolobus solfataricus P2 is able to metabolize n-propanol as the sole carbon source. An average n-propanol consumption rate of 9.7 and 3.3 mg/L/hr was detected using GC-MS analysis from S. solfataricus cultures grown in 0.40 and 0.16% w/v n-propanol, respectively. The detection of propionaldehyde, the key intermediate of n-propanol degradation, produced at a rate of 1.3 and 1.0 mg/L/hr in 0.40 and 0.16% w/v n-propanol cultures, further validated the ability of S. solfataricus to utilize n-propanol. The translational and transcriptional responses of S. solfataricus grown on n-propanol versus glucose were also investigated using quantitative RT-PCR and iTRAQ approaches. Approximately 257 proteins with g2 MS/MS spectra were identified and quantified via iTRAQ. The global quantitative proteome overview obtained showed significant up-regulation of acetyl-CoA synthetases, propionylCoA carboxylase, and methylmalonyl-CoA mutase enzymes. This led to the proposition that the propionyl-CoA formed from n-propanol degradation is catabolised into the citrate cycle (central metabolism) via succinyl-CoA intermediates. In contrast, evidence obtained from these analysis approaches and in vivo stable isotope labeling experiments, suggests that S. solfataricus is only capable of converting isopropyl alcohol to acetone (and vice versa) but lacks the ability to further metabolize these compounds. Keywords: Sulfolobus solfataricus P2 • iTRAQ • alcohol dehydrogenase • shotgun proteomics • alcohol metabolism • stable isotope labeling
Introduction The ability of Sulfolobus solfataricus P2 to grow at high temperature (80 °C) and in acidic environments (pH 2-4) reveals an impressive facility to survive in extreme environments and indicates a possible source of novel industrially relevant enzymes.1 One set of such enzymes are the alcohol dehydrogenases (ADH), with 13 putatively identified from the S. solfataricus P2 genome, using similarity search matrices.2 ADH are enzymes that facilitate the conversion of alcohols to aldehydes or ketones and vice versa. These enzymes are also well-known for their commercial value as chiral alcohol and ketone processing catalysts, especially in ethanol production via biomass fermentation.3 They can be categorized according to their cofactor specificity, which include: (i) nicotinamide * To whom correspondence should be addressed. Prof. Phillip C. Wright, Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK. Tel, +44(0)114 2227577; Fax, +44(0)114 2227501; E-mail, p.c.wright@ sheffield.ac.uk. † Department of Chemical and Process Engineering. ‡ Current address: Oncology Research Institute, National University of Singapore, Centre of Life Sciences, 28 Medical Drive, #02-14, Singapore 117456. § Current address: Metabolic Engineering & Fermentation Group, Ocean Nutrition Canada, 101 Research Drive, Dartmouth, Nova Scotia, B2Y 4T6, Canada. | Academic Unit of Reproductive and Developmental Medicine.
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adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), (ii) flavin adenine dinucleotide (FAD), and (iii) pyrrolo-quinoline quinine, heme, or cofactor F420 types.4 On the basis of gene annotation, all of the adh genes of S. solfataricus have been characterized as NAD+-dependent ZnADH.2,4 Among these, to date only ADH-10 (SSO2536) has been well characterized as being an alcohol dehydrogenase.5-7 ADH-4 (SSO1300) was recently recharacterized as a D-arabinose-1-dehydrogenase (AraDH) enzyme, involved in arabinose degradation.8 The presence of multiple ADH ORFs indicates the potential for significant alcohol metabolism by this organism. This hypothesis was supported by a previous study, which reported the potential of S. solfataricus to utilize alcohols and ketones such as ethanol, n-propanol, isopropyl alcohol, and acetone as cosubstrates along with glucose.9 This work also suggested that these alcohols were oxidized to form aldehydes or ketones by ADH activity. A similar study also reported that one of the S. solfataricus strains was capable of converting isopropyl alcohol to acetone and vice versa, both in the presence and absence of glucose.10 Yet, despite investigations of ADH-10, alcohol metabolism within S. solfataricus P2 has not been widely studied, with the only other study focusing on phenol degradation by S. solfataricus. In that investigation, 10.1021/pr060575g CCC: $37.00
2007 American Chemical Society
Propanol Metabolism in Sulfolobus solfataricus P2
this archaeon was reported to grow solely on phenol, by oxidization via catechol intermediates.11 This study, on the other hand, focused on discerning the metabolic pathways involved in carbon utilization of threecarbon alcohols, by growing S. solfataricus on either n-propanol or isopropyl alcohol as the sole carbon source. The ability of S. solfataricus to incorporate these alcohols into central metabolism for biomass generation was monitored throughout the growth cycle, whereas alcohol uptake and conversion was monitored via gas chromatography-mass spectrometry (GCMS). The ketone, acetone, was also tested, as it is the primary intermediate of isopropyl alcohol oxidation. Stable isotope labeling was employed to assess carbon substrate incorporation into the amino acid pool. We also sought to measure the organism’s global translational response toward these alcohol substrates via the isobaric tags for relative and absolute quantitation (iTRAQ) approach.12 Finally, quantitative real timePCR was employed to determine the changes in adh gene expression levels, in order to facilitate comparisons between transcriptional and translational responses by this organism.
Materials and Methods The Growth Conditions. Sulfolobus solfataricus P2 (DSM 1617) was grown in a pH 4.0 medium at 80 °C, as described in previous studies with some modifications.13,14 Specifically, cultures were supplemented with 0.04-0.40% w/v of either n-propanol, isopropyl alcohol, or acetone (as the first metabolite of isopropyl alcohol conversion) as the sole carbon source (i.e., in the absence of glucose). S. solfataricus grown separately on 0.40% w/v glucose was used as the control. Triplicate cultures were established for each control and experimental conditions, including blank cultures (negative control), i.e. without inoculum. The growth of S. solfataricus was monitored through optical density (OD) at a wavelength of 530 nm, and cell counting was also carried out using a hemeocytometer slide (Marienfield, Germany) under a microscope (Axiostar Plus, Zeiss, USA). All chemicals used were purchased from SigmaAldrich (Gillingham, Dorset, UK), unless otherwise stated. Substrates Measurement. Two-hundred microliters of medium containing cells (from each control and experimental conditions) and cell-free medium from blank cultures (negative control) were collected for analysis. The solvent concentrations remaining in these cultures were monitored throughout growth using a Finnigan Trace DSQ single Quadrupole GC-MS coupled to an auto-sampler (model AS3000, Thermo Electron, Waltham, USA) fitted with a 30 m × 0.25 mm i.d. × 0.25 µm df stabilwax fused silica column (Thames Restek, Bucks, UK). Hot needle injections with a 1 µL injection volume were employed. Helium was used as carrier gas at a constant flow rate of 1 mL/min. The split/splitless injector was set to 220 °C, running on the split mode with 100 mL/min split flow and 1:100 split ratios. The oven temperature profiles were programmed to trace: (a) isopropyl alcohol or acetone degradation: 40 °C held for 0.5 min, followed by a 30 °C/min ramping up to 200 °C and held at 200 °C for 1 min; and (b) n-propanol degradation: 40 °C held for 0.5 min, followed by a 20 °C/min ramping up to 99 °C and held for 2 min, then ramped up to 200 °C at 50 °C/min and held for 1 min at 200 °C. The GC-MS was operated in the Selected Ion Monitor (SIM) mode with 1.0 SIM width and 50 ms dwell time. SIM masses (m/z) used to detect the degradation of each alcohol or ketone were as follows: (a) isopropyl alcohol or acetone: 29, 43, 45, 57, 58, 69, 70, 71; (b) n-
research articles propanol: 26, 27, 28, 29, 31, 58, 59, 85, 100. An internal standard (heptane) was added to all phenotypes for quantification purposes. Quantitative RT-PCR. Mid-exponentially grown cells were preserved on ice immediately prior to centrifugation at 5000 × g at 4 °C for 10 min and DNA-free RNA was extracted (from freshly harvested cells) using the RNeasy Mini Kit (Qiagen, West Sussex, UK) coupled with an on-column DNAse treatment (Qiagen), according to the manufacturer’s protocol. A total of 155 ng of RNA template was used for cDNA synthesis, using the Quantitect Reverse Transcription kit (Qiagen). Quantitative PCR was performed on an iCycler IQ (Bio-Rad, Hertfordshire, UK), using SYBR Green Jump Start Taq ReadyMix reagent (Sigma) and primers designed in-house targeting 13 adh genes, a maleylacetate reductase (clcE) gene, which is also classified as a iron-containing adh gene,2,4 and a housekeeping gene (23S) (refer to Supporting Information).15 Analysis was carried out in triplicate, and standard calibration curves were generated from a series of serial dilutions (dilution factor of 3) of a DNA pool, consisting of all control and experimental samples. The thermal cycling parameters were set at an initial 95 °C for 3 min followed by 50 cycles of 95 °C for 30 s, an annealing temperature ranged between 60 and 66 °C (depending on the primer pairs used, see Supporting Information) for 30 s and 72 °C for 30 s. Melting curve analysis was performed with a temperature ranging from 95 to 55 °C. All gene expression values were normalized against expression of the housekeeping gene (23S). Isobaric Peptide Labeling. S. solfataricus cells were harvested at the mid-exponential phase by centrifugation at 5000 × g for 15 min. The resulting cell pellet was resuspended in 500 mM TEAB, pH 8.0, and soluble protein was extracted using liquid nitrogen coupled with mechanical cracking, as described previously.13,16 Proteins were recovered from the supernatant by centrifugation at 21 000 × g at 4 °C for 30 min, and the total protein concentration was then determined using the RC DC Protein Quantification assay (Bio-Rad, Hertfordshire, UK), according to the manufacturer’s protocol. As described in a previous study, 100 µg of protein from each phenotype was reduced, alkylated, digested, and labeled with iTRAQ reagents according to manufacturer’s protocol (Applied Biosystems) with minor modifications.12 The protein sample from the glucose control was labeled with 114 iTRAQ reagents, whereas protein samples obtained from two different biological replicate cultures, grown on 0.16% w/v n-propanol, were labeled with reagents 115 and 116, with protein samples obtained from 0.40% w/v n-propanol culture labeled with reagent 117. The dried labeled peptides were then combined and resuspended in 0.1% v/v trifluoroacetic acid (TFA) for C18 cleanup using a C18 Discovery DSC-18 SPE column (100 mg capacity, Supelco, Sigma-Aldrich), to remove the excess iTRAQ labeling reagents, prior to in-gel iso-electric focusing (IEF) peptide fractionation. In-gel IEF Peptide Fractionation. Cleaned and dried peptides were resuspended in 8 M urea and 0.5% v/v Pharmalyte pH 3-10. The peptide mixture was loaded onto pH 3-10 NL IPG strip (Bio-Rad), which was rehydrated overnight with 8 M urea, using the cup loading technique.17 The IEF conditions used in this peptide fractionation were identical to those used for the 2-DE and IEF peptide workflows described in our previous work.13 After IEF peptide separation, the IPG strip was cut into 15 fractions, with 13 fractions of 1 cm in the middle, and 2 fractions of 2 cm each at both the acidic and basic ends Journal of Proteome Research • Vol. 6, No. 4, 2007 1431
research articles of the strip (see Figure A in Supporting Information). Peptides were then eluted from the strips via the elution step for in-gel digestion as detailed elsewhere.13 The recovered elutant was then dried via vacuum concentration. These dried peptide fractions were then resuspended in 0.1% v/v TFA for C18 cleanup to remove urea, prior to tandem mass spectrometric analysis.13 Mass Spectrometry and Data Analysis. Mass spectrometric analysis was performed on a QStar XL Hybrid ESI Quadrupole time-of-flight tandem mass spectrometer, ESI-qQ-TOF-MS/ MS (Applied Biosystems, Framingham, MA; MDS-Sciex, Concord, Ontario, Canada) coupled to an online capillary liquid chromatograph (Famos, Switchos and Ultimate liquid chromatography system from Dionex/LC Packings, Amsterdam, The Netherlands), described previously.16 A 120 min gradient was used for all iTRAQ fractions, and the same mass spectrometer set up was used to perform MS and MS/MS analysis as described previously, with the exception that in this instance, a mass range of 300-1500 m/z was selected for data acquisition.12 Protein identification and quantification was carried out using ProQuant software (version 1.1; Applied Biosystems, MDS-Sciex), whereas ProGroup Viewer software version 1.0.6 was used to group and identify proteins with at least 95% confidence. The search was performed against a “mixed genome database” created in-house, which consists of 17 000 entries.12 Here, the search parameters allowed for a peptide and MS/MS tolerance up to 0.15 and 0.1 Da, respectively; one missed cleavage of trypsin; oxidation of methionine, and cysteine modification of MMTS (methyl methanethiosulfonate). Reflux System. A simple reflux system was set up (see Figure B in Supporting Information), with the intention of collecting volatile intermediates for GC-MS detection. For this particular experiment, S. solfataricus grown on a combination of acetone and glucose was used in an attempt to determine the intermediates from acetone degradation onward, since our previous study had demonstrated the ability of S. solfataricus to convert isopropyl alcohol to acetone in the presence of glucose.9 This experiment setup allowed us to investigate further the possibility of isopropyl alcohol/acetone co-metabolism in this organism. Triplicate cultures were maintained at 80 °C with gentle shaking and connected to a reflux column. Cooling water at 4 °C was continuously supplied into the reflux column to condense the vapor evaporated from the culture. The condensate was collected in a closed flask which was placed on ice. The collected distillate was then injected into the GC-MS for intermediate compound identification. The same GC set up was employed as that detailed previously, with the only exception being that the mass spectrometer was operated in the full scan mode at a selected mass range of 10-500 m/z. Stable Isotope Labeling. Similarly, an in vivo metabolic labeling approach was employed to investigate the possibility of cosubstrate acetone incorporation in S. solfataricus. Three separate cultures were grown on (i) 0.40% w/v acetone and 0.40% w/v glucose, (ii) 0.40% w/v acetone and 0.40% w/v 13C universally labeled glucose, and (iii) 0.40% w/v of 13C universally labeled glucose. All cultures were allowed to grow for at least 7 doubling times to ensure full adaptation to the medium.18 These cultures were then harvested in mid-exponential phase by centrifugation at 5000 × g for 15 min at room temperature. The soluble protein fraction was then extracted from the cell pellet using the B-PER Bacterial Protein Extraction Reagent (Pierce Biotechnology, Rockford, USA), according to the manu1432
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facturer’s protocol. The total soluble protein fraction concentration was measured using the RC DC Protein Quantification Assay (Bio-Rad). Protein extracts (10 µg each) were then mixed in a 1:1 ratio, with a combination of (i) a mixture consisting of 10 µg protein from cultures grown on a combination of 0.40% w/v acetone and 0.40% w/v glucose; and 10 µg protein from culture grown on 0.40% w/v 13C universally labeled glucose, and (ii) 10 µg of protein each from cultures grown on 0.40% w/v acetone and either 0.40% w/v glucose or 13C universally labeled glucose; prior to protein separation using 1D SDSPAGE. The gel size was approximately 7 cm × 7 cm × 1 mm with a 4% stacking gel on top of the 12% resolving gel. The gel bands were subsequently (after excision) trypsin-digested for mass spectrometry analysis as detailed previously.13 The information dependent acquisition (IDA) data generated from this experiments was then submitted to Mascot version 1.6b10 available online (www.matrixscience.com) for protein identification. The adjustable search parameters were set to a MS and MS/MS tolerance of 1.2 and 0.6 Da, respectively. Only a single miss-cleavage of trypsin was allowed. A carbamidomethyl modification of cysteine and methionine oxidation was set as the fixed modification and variable modification, respectively. The search was performed against the current Mass Spectrometry protein sequence DataBase (MSDB; ftp://ftp.ncbi.nih.gov/repository/MSDB/msdb.nam). Only proteins identified with Molecular Weight Search (MOWSE) scores greater than 50 were considered significant.1 Furthermore, the carbon compositions of the peptides identified (based on 12C) were calculated using a composition calculator, which is available online (http://db.systemsbiology.net:8080/proteomicsToolkit/). On the basis of the carbon composition calculated, manual inspection was carried out to identify the m/z shifts between the heavy and light versions of each peptides.18 The theoretical spectra of each peptide was generated using IsoPro 3.0 software (http://members.aol.com/ msmssoft/) based on relative isotope abundance (RIA). The experimental spectra was then compared against the theoretical spectra to predict the RIA of the experimental cultures.
Results and Discussion The Growth Profiles. The hypothesis tested in this study, that 3-carbon alcohols may be metabolized, was postulated on results derived from a previous study, which documented coutilization of either isopropyl alcohol or n-propanol in the presence of glucose by S. solfataricus.9 Hence, in this study, S. solfataricus was grown on either isopropyl alcohol or npropanol as the sole carbon source (in the absence of glucose). From the growth profiles depicted in Figure 1, S. solfataricus was shown to grow in both 0.16% and 0.40% w/v n-propanol, having similar specific growth rates of 0.01 h-1 (refer to the Supporting Information), with no growth observed at a higher concentration of 0.80% (data not shown). However, the cell density yields achieved when grown on 0.16% w/v n-propanol were 1.5 times higher than those produced at 0.40% w/v n-propanol (see Figure 1 and Supporting Information). This observation may be due to a concentration related inhibitory effect of n-propanol and metabolic intermediates that are generated at higher concentrations in the 0.40% w/v npropanol cultures. As can be seen in Figure 1, these specific growth rates were found to be 4-fold lower when compared to glucose control cultures (also refer to Supporting Information), lower cell densities were also noted. This was not surprising, as glucose is an easier carbon source to be catabolised in the
Propanol Metabolism in Sulfolobus solfataricus P2
Figure 1. Growth profiles of S. solfataricus in (4) 0.40% w/v n-propanol, (2) 0.16% w/v n-propanol, (0) 0.04% w/v isopropyl alcohol, (9) 0.40% w/v isopropyl alcohol and ()) 0.40% w/v acetone as the sole carbon source. The growth profiles of both (O) glucose control and (b) n-propanol co-metabolism culture (i.e., grown in combination with glucose and 0.40% w/v npropanol) reproduced from previous study,9 are also shown. A culture with no carbon source (×) was also set up as the negative control. All cultures were grown in triplicate.
cells via the Entner-Doudoroff pathway, generating pyruvate as the precursor for tricarboxylic acid cycle (TCA) energy generation and amino acid biosynthesis.1,19,20 Conversely, there was no sign of growth on either isopropyl alcohol or acetone (Figure 1). A range of isopropyl alcohol and acetone concentrations (0.80 to 0.01% w/v) were attempted, yet the cell density of those cultures decreased throughout incubation (data not shown). These profiles were similar to the profile observed in the negative control culture, in which no carbon source (either alcohols or glucose) was supplied. This observation indicates that both isopropyl alcohol and acetone cannot be assimilated into central metabolism by S. solfataricus P2 to generate biomass, despite their ability to convert isopropyl alcohol to acetone or vice versa in the presence of glucose.9 The ability of S. solfataricus to survive on n-propanol instead of isopropyl alcohol may be due to the thermal stability of the compounds. Specifically, a lower chemical polarity is induced in the covalent bond between the carbon atom and the hydroxyl group (-OH) in isopropyl alcohol, as the polarity is reduced by the presence of methyl groups at both ends of the compound. Similarly, electrons are evenly distributed in isopropyl alcohol due to its stereospecific symmetry. This symmetrical structure provides additional stability to the isopropyl alcohol molecule compared to n-propanol, and thus S. solfataricus would need to use more energy to break these bonds. The stability of isopropyl alcohol is reflected by its higher Gibbs free energy of formation value (∆Gfø ) -185.9 kJ/mol) compared to n-propanol (∆Gfø ) -175.8 kJ/mol) under standard conditions (298 K, 1 atm).21,22 Furthermore, the symmetrical structure of acetone (∆Gfø ) -161.2 kJ/mol) formed from isopropyl alcohol oxidation is also a more stable molecule compared to propionaldehyde (n-propanol oxidation product) (∆Gfø ) -142.1 kJ/mol).22,23 Therefore, it is more thermodynamically feasible for S. solfataricus to successfully incorporate n-propanol/propionaldehyde compared to isopropyl alcohol/ acetone. However, the ability of S. solfataricus to utilize different alcohols also depends very much on the cells’ tolerance toward the solvent toxicity imposed, as well as the physiological features such as the availability of the transport systems. These
research articles factors which can be somewhat negated due to the ability of this organism to survive by co-utilization with glucose, as demonstrated elsewhere.9 Isopropyl Alcohol and Acetone Co-Metabolism? Although our previous study has demonstrated the ability of S. solfataricus to grow on both isopropyl alcohol and acetone, and its capability to degrade isopropyl alcohol to acetone in the presence of glucose,9 results here show that the growth on isopropyl alcohol/acetone as the sole carbon source is not possible, and reveal the inability of this micro-organism to incorporate these compounds into central metabolism for biomass generation. Here, to explore further the potential of this microorganism in incorporating isopropyl alcohol/acetone as a cosubstrate (i.e., in the presence of glucose), a simple reflux system (Figure B in Supporting Information) was developed (see the materials and methods section) to capture and analyze any volatile metabolic intermediates which may previously have been overlooked. Yet, using GC-MS analysis only acetone and isopropyl alcohol were detected in the resulting condensate collected. No other intermediate such as acetoacetate, the next step in acetone metabolism, was detected. Similar findings were obtained when an identical culture was frozen in liquid nitrogen immediately upon cessation of fermentation, in order to preserve any possible intermediates, prior to GC-MS analysis (data not shown). These findings were not surprising as intermediate compounds such as acetoacetate are very unstable. Furthermore, a stable isotope labeling technique was also employed to further verify the hypothesis that isopropyl alcohol and/or acetone co-incorporation exists within S. solfataricus, because this micro-organism is unable to survive solely on isopropyl alcohol or acetone, and yet these solvents (isopropyl alcohol and acetone) were consumed in the presence of glucose (described previously).9 The cultures were fully adapted to the media in order to obtain ∼100% labeling efficiency. Here, the labeling efficiency is defined as the ratio between the area of labeled peptide (heavy isotope) and the unlabeled peptide (light isotope).18 The incorporation efficiency, conversely, is defined as the ratio between the number of 13C atoms with respect to the total number of carbons (12C and 13C) and is dependent upon the RIA of the medium.18 For example, a fully adapted culture will give an incorporation efficiency that equals the RIA of the medium. In this study, the combination of 13C labeled glucose and 12C acetone supplemented into the media corresponded to a RIA of 39.5% 13C or 60.5% 12C. Therefore if acetone was co-incorporated in this fully adapted culture, the experimental peptide mass spectrum distribution should be relatively similar to the theoretical spectral distribution calculated using the IsoPro software and having a RIA value of 39.5% 13 C incorporation. However, all experimental peptide distributions obtained corresponded to a theoretical spectral distribution with a RIA of 98.9% 13C incorporation. For instance, Figure 2 illustrates an example of both the theoretical and experimental spectra obtained from the doubly charged peptide (FVPVVAPAGDNITHR) identified from hypothetical protein TuF-1 (SSO0216). The experimental peptide distribution obtained from cultures grown in 12C (acetone and glucose) and 13C glucose (in the absence of acetone) were comparable to a theoretical RIA of 98.9% 12C and 98.9% 13C incorporation respectively (see Figure 2D). The ratio of the peptide areas generated from these two cultures was approximately 1.18, which was reasonable, as proteins from these two cultures were mixed in a 1:1 ratio. Similarly, a 12C acetone Journal of Proteome Research • Vol. 6, No. 4, 2007 1433
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Figure 2. (A-C) Theoretical spectral distribution calculated from IsoPro 3.0 software (http://members.aol.com/msmssoft/) on the basis of a carbon RIA for peptide sequence FVPVVAPAGDNITHR identified from hypothetical protein TuF-1 (SSO0216), whereas the experimental spectral of this peptide obtained from the mixture of different cultures is also illustrated, including the protein mixture obtained from the combination of (D) cultures grown in 12C (acetone and glucose) and those grown in 13C glucose and (E) cultures grown in 12C (acetone and glucose) and those grown in a mixture of 12C acetone and 13C glucose. The experimental spectral distribution obtained from cultures grown in a mixture of 12C acetone and 13C glucose (E(iv)) was found to be similar to the theoretical spectral having a 13C RIA of 98.9%. This suggests no incorporation of 12C from any acetone sources supplemented into the culture.
and 13C glucose culture also gave a ratio of approximately 1.20 when compared to cultures grown in 12C (acetone and glucose). The peptide mass spectra of cultures grown on the mixture of 12 C acetone and 13C glucose showed a RIA for 13C incorporation of 98.9%, which was similar to that obtained from culture grown solely on 13C glucose (Figure 2). This spectral distribution indicates that only 13C from glucose was incorporated by S. solfataricus, with no sign of 12C (acetone) incorporation observed. This same observation was consistent throughout all peptides spectra obtained in this study. Thus, this evidence suggests that S. solfataricus may convert isopropyl alcohol to acetone (or vice versa) by incorporating a hydrogen molecule for NADH generation, yet no further incorporation of acetone was observed. Future studies will focus on in vitro assay measurement of the acetone carboxylation reaction to further explore the potential of acetone catabolism in this microorganism, as was carried out in other bacteria such as Xanthobacter strain PY2, Rhodococcus rhodochrous B276 (ATCC 31338), and Desulfococcus biacutus strain KMRActS.24-26 n-Propanol Consumption. Conversely, the significant growth (Figure 1) and utilization profiles (Figure 3) of n-propanol proves that S. solfataricus is indeed able to assimilate n1434
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propanol as sole carbon sources for biomass generation. Specifically, a higher average n-propanol consumption rate of 9.7 mg/L/hr was observed in the 0.40% w/v n-propanol culture (approximately 3-fold higher), compared to 3.3 mg/L/hr in 0.16% w/v n-propanol. Interestingly, the consumption rate of n-propanol in the 0.40% w/v cultures was also found to be 3 times higher than that the cultures grown in 0.40% w/v n-propanol with the presence of glucose (at 3.3 mg/L/hr, demonstrated in previous study9). In addition, the propionaldehyde intermediate was detected via GC-MS at a production rate of 1.3 and 1.0 mg/L/hr in 0.40 and 0.16% w/v n-propanol cultures, respectively. The accumulation of propionaldehyde from n-propanol oxidation in the growth media (containing cells) supports the hypothesis of n-propanol degradation by S. solfataricus. It is still unclear about the subsequent processes or the bio-degradation products formed (from n-propanol degradation) that were eventually incorporated or transported into the cells. Expression of ADHs at the Protein and Transcript Levels. The oxidation of n-propanol to propionaldehyde is catalyzed by alcohol dehydrogenase enzymes and results in the reduction reducing nicotinamide adenine dinucleotide (NAD+). By taking
Propanol Metabolism in Sulfolobus solfataricus P2
Figure 3. Average n-propanol utilization profiles of S. solfataricus cultures grown on (2) 0.16% w/v and (9) 0.40% w/v of n-propanol (in the absence of glucose). GC-MS analysis determined that propionaldehyde was formed from the oxidation of n-propanol, and their accumulation profiles are given using unfilled data points for both (4) 0.16% and (0) 0.40% w/v n-propanol cultures, respectively. These average values were obtained from triplicate cultures which had taken solvent evaporation into account.
Figure 4. Gene and protein expression of ADHs identified in S. solfataricus grown in 0.16 and 0.40% w/v n-propanol compared to the glucose control. These transcriptional and translational results were obtained from quantitative RT-PCR and iTRAQ approaches, respectively.
both the biological and technical variations into consideration, i.e., using a ( 50% variation cutoff point in iTRAQ approach,27 the putative alcohol dehydrogenase, ADH-2 was found to be up-regulated at least 1.7-fold in both 0.16 and 0.40% w/v n-propanol cultures compared to the glucose control at the protein level (refer to Figure 4). Furthermore, the putative alcohol dehydrogenases, ADH-5 (1.7-fold) and ADH-12 (2.8fold) were up-regulated when grown on 0.40% w/v n-propanol but remained unchanged in 0.16% w/v n-propanol cultures. At the transcript level, on the other hand, adh-2 was found to be 2.5-fold up-regulated in 0.16% w/v n-propanol cultures compared to glucose control, yet remained unchanged in 0.40% w/v n-propanol. Conversely, the transcription levels of adh-5 and adh-12 showed no significant changes in 0.16% w/v n-propanol and were actually down-regulated by >2.5-fold in 0.40% w/v n-propanol cultures. The discrepancies between the transcript and protein expression levels observed here suggested that protein expression level may be influenced by various factors, apart from the amount of mRNA present, including protein turnover and post-
research articles translational modifications, as reported elsewhere.28,29 The expression level of the genes and proteins also depends on the rate of the synthesis, degradation, and accumulation.30 The mRNA molecules are relatively unstable compared to proteins in general, contributing to the difference in turnover rates between mRNA and protein, as reported in Saccharomyces cerevisiae31 and Escherichia coli.32 The result being that the different half-lives of proteins and genes also contributes to the deviation in the expression level (at the same sampling time point). The half-life of protein is reported to be longer compared to the gene.29 For example, it has been reported that the median S. solfataricus mRNAs half-life is 5 min,33 whereas a half-life of 3-8 min for E. coli mRNAs has been determined.34 In contrast, Brouns et al. report that the R-galactosidase enzyme of S. solfataricus shows a half-life of 30 min.35 Predictions of the median half-life of 3751 proteins measured in S. cerevisiae yields a value of approximately 43 min, significantly longer than mRNAs half-lives.36 Interestingly, from the transcriptional data (refer to Supporting Information), adh-1 and adh-10 were also found to be highly up-regulated (>4-fold) in all n-propanol cultures; yet these alcohol dehydrogenases could not be identified at the protein level. The expression levels of several adh genes were also found to be significantly different compared to glucose control cultures. Here, the expression levels of adh-8, adh-9, and clcE genes were found to be down-regulated at least 1.7-fold. A change in the expression level of adh-4 was also found to be minimal in this study, which is not surprising, because this gene has been reannotated as a D-arabinose-1dehydrogenase (AraDH) gene, involved in the arabinose pathway.8 Fiorentino et al. revealed that there are multiple factors and control elements that contribute to the regulation of adh genes transcription in S. solfataricus.37 The study identified a functional transcriptionally active sequence in the 5′ flanking region of the an alcohol dehydrogenase gene (Ssadh, ADH-10, SSO2536) in this organism, and reported binding sites for DNA binding proteins (Sso7d and Sso10b (Alba)), and transcription factors (Lrs14). The molecular mechanism regulating the adh genes transcription was also well-studied.37 However, they proposed that the identified regulatory site is unique to a specific set of functions, without sharing with other putative adh genes annotated in the genome. Proteome Analysis of n-Propanol Metabolism. The overview of n-propanol assimilation into the central metabolic pathway of S. solfataricus was demonstrated in this study via the global proteome regulation values obtained using iTRAQ. Approximately 257 proteins with g2 MS/MS spectra were identified and quantified via the iTRAQ approach (Table 1). As mentioned previously, by taking a cutoff point of ( 50% variation (taking into account technical, experimental, and biological replicates),27 18% of the proteins identified were found to be upregulated (g1.5-fold) compared to the glucose control. Similarly, an average of 18% of the proteins identified were found to be down-regulated, with 64% remaining unchanged. The complete list of protein identifications obtained in this study is tabulated in Supporting Information, and also available from our website (http://wrightlab.group.shef.ac.uk/projects/index.htm). Specifically, we propose that n-propanol was incorporated into central metabolism via the production of propionyl-CoA intermediates which further degraded to succinyl-CoA, a main TCA cycle intermediate (as shown in Figure 5).38 There are a Journal of Proteome Research • Vol. 6, No. 4, 2007 1435
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Table 1. Total Number of Proteins and Their Regulation Obtained via the iTRAQ Approacha total protein identifications proteins with g2 MS/MS spectra
396 257
Protein distribution (%)b protein regulation
up (ratio g 1.50) unchanged (0.66 < ratio < 1.50) down (ratio e 0.66)
0.16% w/v n-propanolc
0.16% w/v n-propanolc
0.40% w/v n-propanol
12.8
19.1
22.2
69.3
70.4
53.7
17.9
10.5
24.1
a By taking into account biological and technical variations, the change in protein regulation was considered significant if at least ( 50% variation was noted compared to the glucose control. b Only those proteins identified with g2 MS/MS spectra were taken into account. c From different biological samples.
number of pieces of evidence that lend support to this, metabolite and proteomic, which are described in the following paragraphs. As mentioned previously, the initial degradation reaction is the oxidation of n-propanol by alcohol dehydrogenases to form propionaldehyde (Figure 5). The propionaldehyde formed is then oxidized to propanoic acid by reducing NAD+. However, protein expression results showed that the putative aldehyde dehydrogenase, SSO3117, annotated for the aldehyde oxidation reaction, remained unchanged in 0.16% w/v n-propanol cultures and exhibited a 2-fold down-regulation in 0.40% w/v n-propanol cultures (refer to Table 2). This is due to the presence of an incorrectly annotated enzyme (SSO3117), now characterized as a 2,5-dioxopentanoate dehydrogenase (DopDH), within this alcohol degradation pathway.8 Hence, there remains a missing step in the monitoring of all enzymes involved in the oxidation of n-propanol into the TCA cycle. There are also several uncharacterised, putative glyceraldehyde dehydrogenases (including SSO1629, SSO1842, and SSO1218) in S. solfataricus, which may be potentially involved in npropanol degradation. For example, the glyceraldehyde-3phosphate dehydrogenase (SSO1842) was found to be >1.8fold up-regulated in 0.40% w/v n-propanol cultures compared to the control cultures (see Supporting Information). Propanoic acid, formed through the oxidation of propionaldehyde, is further catalyzed by the acetyl-CoA synthetases (SSO2863 and SSO3203) to produce propionyl-CoA via a propionyl adenylate intermediate, as shown in Figure 5. To provide evidence of this route, these acetyl-CoA synthetases were found to be >1.5-fold up-regulated in n-propanol cultures, when compared to the control. The propanoic acid may also be directly converted to produce propionyl-CoA (without forming propionyl adenylate intermediate) by acetyl-CoA synthetases, as proposed in Salmonella enterica serovar Typhimurium LT2.39 Propionyl-CoA carboxylase (SSO2463), on the other hand, showed an up-regulation of >1.8-fold in the 0.16% w/v npropanol culture, but remained constant in 0.40% w/v npropanol (as tabulated in Table 2). In addition, biotin carboxylase, a subunit of propionyl-CoA carboxylase (SSO2466), was also found to be at least 1.5-fold up-regulated in 0.16% w/v n-propanol cultures. Propionyl-CoA carboxylase is a biotin enzyme which has the same catalytic mechanism as acetylCoA carboxylase and pyruvate carboxylase.38,40-43 This enzyme 1436
Journal of Proteome Research • Vol. 6, No. 4, 2007
Figure 5. Possible, simplified n-propanol metabolism pathway for S. solfataricus. The annotated genes involved in this pathway were obtained from KEGG (http://www.genome.jp/kegg/ kegg2.html) and Snijders et al.1 Some steps currently have no enzymes annotated (N/A). Regulation of proteins determined by iTRAQ analysis are represented by: (v) up-regulated, (≈) no change, and (V) down-regulated in either 0.16 or 0.40% w/v n-propanol cultures compared to glucose control, whereas (×) indicates that the proteins were not identified in this study.
carboxylates propionyl-CoA by consuming ATP to yield (S)methylmalonyl-CoA, which is then isomerized into (R)-methylmalonyl-CoA. The (R)-methylmalonyl-CoA acts as the substrate for a vitamin B12 dependent mutase that converts this isomer into succinyl-CoA.38,44 This scenario is supported by empirical data showing an up-regulation (>1.5-fold) of methylmalonyl-CoA mutase, alpha-subunit (SSO2425) in the 0.16% w/v n-propanol cultures. The generation of succinyl-CoA through a series of reactions from n-propanol breakdown is then synthesized via TCA cycle. A similar pathway was also proposed in the propionate metabolism of Pseudomonas butanovora, where propionate is utilized and proceeded via propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA into TCA cycle.45 Leal et al.46 and Berg et al.47 also reported the comparable pathway of propionyl-CoA degradation into TCA cycle in S. enterica serovar Typhimurium LT2 and Rhodospirillum rubrum, respectively. Besides the pathway discussed above, propanoic acid generated from n-propanol degradation (or even the oxidation of fatty acids that have odd numbers of carbon atoms), may be converted into propionyl-CoA via an acryloyl-CoA intermediate (refer to Figure 5). In this alternative pathway, propanoic acid is converted to malonate semialdehyde and then to 3-hydroxypropanoate, yet so far no enzyme has been annotated for either of these reactions. The 3-hydroxy-propanoate intermediate formed may then be converted to 3-hydroxy-propionyl-CoA by a medium chain fatty acid-CoA ligase (SSO2875). However, protein expression of these annotated enzymes remained
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Propanol Metabolism in Sulfolobus solfataricus P2
Table 2. Identification and Quantification of the Annotated Proteins Involved in Central Metabolism and n-Propanol Degradationa
accession
SSO0764 SSO1646 SSO2800 SSO3117 SSO2863 SSO3203 SSO2463 SSO2425 SSO2875 SSO0654 SSO2514 TCA Cycle SSO2589 SSO1095 SSO2182 SSO2815 SSO2482 SSO2483 SSO2356 SSO2357 SSO2589 SSO1333 SSO1334 SSO3197 SSO2639 SSO0207 SSO2281 SSO0286 SSO0528 SSO0527 SSO0913 SSO0883 SSO2869 SSO2466 SSO1907 SSO1930 SSO2044 SSO0897 SSO2427 SSO0962 SSO0250 SSO0999
0.40%w/v n-propanol
0.16% w/v n-propanold
0.16% w/v n-propanold
Log ratiob
Stdevc
Log ratiob
Stdevc
Log ratiob
Stdevc
8 2 3 5 9 2 2 2
0.34 -0.26 -0.04 -0.11 0.22 0.17 0.25 0.20
0.08 0.00 0.11 0.01 0.07 0.26 0.01 0.04
0.37 0.03 0.03 -0.12 0.35 0.24 a0.41 0.21
0.06 0.05 0.02 0.02 0.04 0.10 0.02 0.02
0.22 0.24 0.45 -0.29 0.31 0.44 -0.02 0.11
0.06 0.04 0.36 0.04 0.09 0.35 0.10 0.01
3 6 3
-0.01 -0.04 -0.30
0.22 0.02 0.05
0.10 -0.08 -0.04
0.17 0.01 0.04
-0.10 -0.05 -0.08
0.15 0.03 0.16
citrate synthase 4.1.3.7 5 0.01 aconitate hydratase 4.2.1.3 11 -0.03 (idh) isocitrate dehydrogenase, probable 1.1.1.42 7 -0.08 2-oxoacid-ferredoxin oxidoreductase, alpha chain 1.2.7.16 -0.19 (sucD) succinyl-CoA synthetase, alpha subunit 6.2.1.5 3 -0.14 (sucC) succinyl-CoA synthetase, beta subunit 6.2.1.5 8 -0.10 (sdhA) succinate dehydrogenase subunit A 1.3.99.1 2 -0.01 (sdhB) succinate dehydrogenase subunit B 1.3.99.1 10 0.03 citrate synthase 4.1.3.7 5 0.01 glyoxylate cycle (aceA/icl) isocitrate lyase 4.1.3.1 8 0.20 (aceB/mas) malate synthase, putative 4.1.3.2 11 -0.14 glycolysis/gluconeogenesis (eda) 2-keto-3-deoxy gluconate aldolase 4.1.2.2 -0.04 (cutA-4) carbon monoxide dehydrogenase, 1.2.99.2 5 0.30 large chain (pmM) phosphomannomutase 5.4.2.8 11 0.04 conserved hypothetical protein 2 0.03 fructose-biphosphatase [46] 10 0.14 (gap) glyceraldehyde-3-phosphate 1.2.1.12 6 0.01 dehydrogenase (GAPDH) phosphoglycerate kinase 2.7.2.3 2 0.06 enolase 4.2.1.11 2 -0.29 (ppsA-1) phosphoenolpyruvate synthase 2.7.9.2 17 0.19 other metabolisms linking out from TCA cycle NAD-dependent malic enzyme 1.1.1.8 0.00 (malate oxidoreductase) (accC) biotin carboxylase a subunit of 6.3.4.14 4 0.19 propionyl-CoA carboxylase (gdhA-2) NAD specific glutamate dehydrogenase 1.4.1.3 3 -0.40 (gdhA-3) NAD specific glutamate dehydrogenase 1.4.1.3 4 -0.10 (gdhA-4) NAD specific glutamate dehydrogenase 1.4.1.3 2 -0.59 (aspB-2) aspartate aminotransferase 2.6.1.1 4 -0.30 other proteins small heat shock protein hsp20 family 5 0.39 DNA binding protein SSO10b 31 -0.14 (radA) DNA repair protein radA 6 -0.06 ABC transporter 5 0.54
0.02 0.05 0.02 0.03 0.14 0.02 0.11 0.01 0.02
-0.10 0.08 -0.09 -0.14 0.00 -0.03 0.11 0.07 -0.10
0.07 0.03 0.02 0.02 0.02 0.02 0.09 0.01 0.07
-0.22 0.07 -0.03 -0.15 0.04 -0.01 -0.14 0.02 -0.22
0.16 0.05 0.03 0.04 0.08 0.12 0.03 0.09 0.16
0.07 0.03
0.17 0.12
0.09 0.02
0.18 0.23
0.13 0.04
0.16 0.06
-0.04 0.29
0.15 0.07
0.19 0.39
0.26 0.07
0.07 0.01 0.03 0.02
0.16 0.20 0.12 0.04
0.04 0.00 0.04 0.01
0.13 -0.11 0.08 0.00
0.08 0.01 0.07 0.05
0.02 0.13 0.06
-0.58 -0.29 0.18
0.03 0.26 0.04
0.07 -0.19 0.21
0.05 0.18 0.05
0.07
-0.09
0.10
0.12
0.05
0.06
0.19
0.06
0.16
0.06
0.22 0.05 0.07 0.07
-0.51 -0.02 -0.62 -0.05
0.20 0.04 0.33 0.09
-0.78 -0.20 -0.48 -0.04
0.05 0.06 0.19 0.09
0.09 0.02 0.06 0.03
0.28 -0.12 0.31 0.29
0.05 0.01 0.02 0.03
-0.15 -0.19 0.14 0.22
0.15 0.04 0.04 0.09
protein name
EC
no. of MS/MS
n-propanol degradation (adh-2) alcohol dehydrogenase (Zn containing) 1.1.1.1 (adh-5) alcohol dehydrogenase (Zn containing) 1.1.1.1 (adh-12) alcohol dehydrogenase (Zn containing) 1.1.1.1 (aldhT) aldehyde dehydrogenase 1.2.1.3 (acsA-9) acetyl-CoA synthetase 6.2.1.1 (acsA-10) acetyl-CoA synthetase 6.2.1.1 (ppcB) propionyl-CoA carboxylase beta subunit 6.4.1.3 (mcmA1) methylmalonyl-CoA mutase, 5.4.99.2 alpha-subunit, chain A (alkK-4) medium-chain-fatty-acid-CoA ligase 6.2.1.(paaF-2) enoyl CoA hydratase 4.2.1.17 3-hydroxyacyl-CoA dehydrogenase/ 1.1.1.157/ enoyl CoA hydratase 4.2.1.17
a The changes in protein regulation were only considered significant with at least ( 50% variation compared to a glucose control culture (taking into account the variation in biological and technical replicates). Therefore, proteins were considered (i) up-regulated when the log ratio is g0.18 and down regulated when log ratio is e-0.18. b The log average ratio is calculated from the list of peptides identified for the proteins. c Stdev refers to standard deviation. d Different biological cultures.
unchanged in n-propanol cultures when compared to the glucose control, signifying this pathway is not especially important under these conditions (Table 2). Similarly, the relative protein abundances of enoyl-CoA hydratase (SSO0654) and 3-hydroxyacyl-CoA dehydrogenase (SSO2514), which are annotated to convert 3-hydroxy-propionyl-CoA to acryloyl-CoA before forming propionyl-CoA, were found to be unchanged. Therefore, this alternate pathway is less likely to be strongly
active here, as most of the annotated proteins along this pathway had no significant change in relative abundance compared to glucose control. Furthermore, the incomplete annotation of this pathway may also suggest a rudimentary presence and limited probable activity in S. solfataricus. So far, the postulated pathway is hypothetical and detailed studies to reveal the n-propanol pathway including the dynamic tracing or steady state analysis using either nuclear Journal of Proteome Research • Vol. 6, No. 4, 2007 1437
research articles magnetic resonance (NMR) or GC-MS via analysis of derivatized proteinogenic amino acids and enzymatic assay of cell lysates from different cultures (glucose and n-propanol cultures) are the subjects of future work. This is also the first and preliminary evidence of the hitherto unknown propanol metabolism reported in S. solfataricus. It would be interesting to further explore and compare the potential of other Sulfolobus strains in assimilating propanol into central metabolism, since similar protein annotations were also found in the S. acidocaldarius and S. tokodaii genomes. Apart from the protein regulation noted along the n-propanol degradation pathway, the majority of the proteins identified using the iTRAQ approach related to the TCA cycle had no significant change in their relative abundance compared to the glucose control. This signifies the presence of comparable central metabolic activities in these n-propanol cultures to that of the glucose control. Nonetheless, there was an up-regulation by >1.5-fold in isocitrate lyase (SSO1333) and malate synthase (SSO1334) proteins in 0.40% w/v n-propanol cultures, which are involved in the glyoxylate cycle. The glyoxylate cycle is generally the metabolic reaction path where enzymes convert twocarbon acetyl units into four-carbon succinate units, and glyoxylate for energy production and biosynthesis by bypassing two decarboxylation steps in the TCA cycle.48,49 The succinate produced is then channelled into the TCA cycle to form malate, whereas glyoxylate will condense in the presence of acetyl-CoA to form oxaloacetate. Both of these intermediates (malate and oxaloacetate) can subsequently be converted into phosphoenolpyruvate for ultimate glycogen production via gluconeogenesis.1,19,20 The up-regulation of >1.5-fold in biotin carboxylase (SSO2466) and phosphoenol-pyruvate synthase (SSO0883) obtained in the n-propanol grown cultures indicates that phosphoenol-pyruvate was formed from oxaloacetate and pyruvate. The proteins annotated to convert 2-oxoglutarate to glutamate or vice versa, including glutamate dehydrogenase (SSO1907, SSO2044), were also found to be >2.5-fold down-regulated in all n-propanol cultures. This down-regulation in protein expression may be due to the lower growth and biomass generation properties of n-propanol cultures compared to the glucose control cultures, as discussed previously.50 The cells may exist under stress conditions and the assimilation of n-propanol into biomass is therefore less efficient.38,51 This scenario was supported by the identification of a 2-fold upregulation in the heat shock protein (SSO2427), in 0.16% w/v n-propanol (refer to Table 2).
Conclusions S. solfataricus has the potential to catabolise n-propanol as the sole carbon source, despite having a 4-fold lower growth rate when compared to the glucose control culture. An average of 9.7 and 3.3 mg/L/hr of n-propanol utilization were obtained in cultures grown on 0.40 and 0.16% w/v n-propanol, respectively. The primary degradation product, propionaldehyde, was also detected during n-propanol degradation and was accumulated at the rate of approximately 1.3 or 1.0 mg/L/hr in 0.40 and 0.16% w/v n-propanol cultures, respectively. Conversely, S. solfataricus P2 showed no signs of growth in culture supplied with either isopropyl alcohol or acetone, in the absence of glucose. Various experiments, including a simple reflux system and in vivo metabolic labeling, suggested that this organism is only capable of converting isopropyl alcohol to acetone in the presence of glucose or vice versa; however, no further catabolism of acetone was observed. An in depth 1438
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investigation employing in vitro enzyme assay measurements will be the subject of future work to further explore the possibility of acetone catabolism in S. solfataricus. Furthermore, 18% (on average) of the 247 proteins identified with g2 MS/MS spectra via an iTRAQ workflow were found to be up- and down-regulated, whereas 64% remained unchanged in n-propanol cultures compared to the glucose control. Among these identified proteins, ADH-2 was found to be up-regulated (>1.7-fold) in n-propanol cultures. This finding correlated well with up-regulation values obtained at the transcript level via quantitative RT-PCR and suggests an important role in npropanol oxidation. Furthermore, global proteome quantitative analysis also showed significant up-regulation in enzymes involved in a hypothesised n-propanol degradation pathway, including acetyl-CoA synthetases (SO2863 and SSO3203), propionyl-CoA carboxylase (SSO2463) and methylmalonyl-CoA mutase (SSO2425). The up-regulation of these enzymes suggests that n-propanol was incorporated into central metabolism via the formation of a succinyl-CoA intermediate, which was further catabolised into the citrate cycle at levels similar to those of the glucose control.
Acknowledgment. P.C.W. thanks the EPSRC for provision of an Advanced Research Fellowship (GR/A11311/01) and for funding (GR/S84347/01). P.K.C. acknowledges the Overseas Research Students Award Scheme (ORSAS). We thank Chee Sian Gan, Martin Barrios-Llerena, Bram Snijders, Aristophanes Stephen Georgiou, and Sarah Elliot for technical support. Supporting Information Available: Figures A and B and Tables. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Snijders, A. P.; Walther, J.; Peter, S.; Kinnman, I.; de Vos, M. G.; van de Werken, H. J.; Brouns, S. J.; van der Oost, J.; Wright, P. C. Proteomics 2006, 6 (5), 1518-1529. (2) She, Q.; Singh, R. K.; Confalonieri, F.; Zivanovic, Y.; Allard, G.; Awayez, M. J.; Chan-Weiher, C. C.; Clausen, I. G.; Curtis, B. A.; De Moors, A.; Erauso, G.; Fletcher, C.; Gordon, P. M.; Heikampde Jong, I.; Jeffries, A. C.; Kozera, C. J.; Medina, N.; Peng, X.; ThiNgoc, H. P.; Redder, P.; Schenk, M. E.; Theriault, C.; Tolstrup, N.; Charlebois, R. L.; Doolittle, W. F.; Duguet, M.; Gaasterland, T.; Garrett, R. A.; Ragan, M. A.; Sensen, C. W.; Van der Oost, J. Proc. Natl. Acad. Sci. U.S.A 2001, 98 (14), 7835-40. (3) Lamed, R. J.; Zeikus, J. G. Biochem. J. 1981, 195 (1), 183-190. (4) Radianingtyas, H.; Wright, P. C. FEMS Microbiol. Rev. 2003, 27 (5), 593-616. (5) Ammendola, S.; Raia, C. A.; Caruso, C.; Camardella, L.; D’Auria, S.; De Rosa, M.; Rossi, M. Biochemistry 1992, 31 (49), 1251412523. (6) Cannio, R.; Fiorentino, G.; Rossi, M.; Bartolucci, S. FEMS Microbiol. Lett. 1999, 170 (1), 31-39. (7) Cannio, R.; Fiorentino, G.; Carpinelli, P.; Rossi, M.; Bartolucci, S. J. Bacteriol. 1996, 178 (1), 301-305. (8) Brouns, S. J.; Walther, J.; Snijders, A. P.; van de Werken, H. J.; Willemen, H. L.; Worm, P.; de Vos, M. G.; Andersson, A.; Lundgren, M.; Mazon, H. F.; van den Heuvel, R. H.; Nilsson, P.; Salmon, L.; de Vos, W. M.; Wright, P. C.; Bernander, R.; van der Oost, J. J. Biol. Chem. 2006, 281 (37), 27378-27388. (9) Chong, P. K.; Burja, A. M.; Radianingtyas, H.; Fazeli, A.; Wright, P. C. Proteomics 2006, in press. (10) Radianingtyas, H.; Wright, P. C. Biotechnol. Lett. 2003, 25 (7), 579583. (11) Izzo, V.; Notomista, E.; Picardi, A.; Pennacchio, F.; Di Donato, A. Res. Microbiol. 2005, 156 (5-6), 677-689. (12) Chong, P. K.; Gan, C. S.; Pham, T. K.; Wright, P. C. J. Proteome Res. 2006, 5 (5), 1232-1240. (13) Chong, P. K.; Wright, P. C. J. Proteome Res. 2005, 4 (5), 17891798. (14) Snijders, A. P. L.; De Vos, M. G. J.; Wright, P. C. J. Proteome Res. 2005, 4 (2), 578-585.
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Journal of Proteome Research • Vol. 6, No. 4, 2007 1439
