Proteome Reference Map and Comparative Proteomic Analysisbetween a Wild Type Clostridium acetobutylicum DSM 1731 and its

Mutant with Enhanced Butanol Tolerance and Butanol Yield

Shaoming Mao,†,‡,§ Yuanming Luo,†,‡,| Tianrui Zhang,‡ Jinshan Li,‡ Guanhui Bao,‡,§ Yan Zhu,‡,§

Zugen Chen,⊥ Yanping Zhang,‡ Yin Li,*,‡ and Yanhe Ma‡,|

Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, Graduate School of Chinese Academy ofSciences, Beijing, China, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese

Academy of Sciences, Beijing, China, and Department of Human Genetics, School of Medicine, University ofCalifornia, Los Angeles, California 90095

Received December 29, 2009

The solventogenic bacterium Clostridium acetobutylicum is an important species of the Clostridiumcommunity. To develop a fundamental tool that is useful for biological studies of C. acetobutylicum,we established a high resolution proteome reference map for this species. We identified 1206 spotsrepresenting 564 different proteins by mass spectrometry, covering approximately 50% of majormetabolic pathways. To better understand the relationship between butanol tolerance and butanolyield, we performed a comparative proteomic analysis between the wild type strain DSM 1731 and themutant Rh8, which has higher butanol tolerance and higher butanol yield. Comparative proteomicanalysis of two strains at acidogenic and solventogenic phases revealed 102 differentially expressedproteins that are mainly involved in protein folding, solvent formation, amino acid metabolism, proteinsynthesis, nucleotide metabolism, transport, and others. Hierarchical clustering analysis revealed thatover 70% of the 102 differentially expressed proteins in mutant Rh8 were either upregulated (e.g.,chaperones and solvent formation related) or downregulated (e.g., amino acid metabolism and proteinsynthesis related) in both acidogenic and solventogenic phase, which, respectively, are only upregulatedor downregulated in solventogenic phase in the wild type strain. This suggests that Rh8 cells haveevolved a mechanism to prepare themselves for butanol challenge before butanol is produced, leadingto an increased butanol yield. This is the first report on the comparative proteome analysis of a mutantstrain and a base strain of C. acetobutylicum. The fundamental proteomic data and analyses will beuseful for further elucidating the biological mechanism of butanol tolerance and/or enhanced butanolproduction.

Keywords: Clostridium acetobutylicum • proteome reference map • comparative proteome analysis •two-dimensional gel electrophoresis • acidogenesis • solventogenesis • butanol tolerance • butanol yield

Introduction

Clostridium acetobutylicum is a low-GC-content, Gram-positive, spore-forming, obligate anaerobe that is capable offermenting a wide variety of sugars (e.g., glucose, galactose,cellobiose, mannose, xylose, and arabinose) to acids (acetic acidand butyric acid) and solvents (acetone, butanol, and ethanol).1

C. acetobutylicum culture was extensively used to produceacetone and butanol from starch for industrial purposes.2

Recently, anaerobic fermentative production of butanol usingthis bacterium gained remarkable interest as butanol is con-sidered as a potential superior biofuel alternative.3 Recentdevelopments in genetics,4 genomics,5 and proteomics6,7 of C.acetobutylicum have greatly increased our understanding of thesolvent production physiology, which is extremely importantfor improving butanol production by means of metabolicengineering or systems biotechnology.

Butanol toxicity is the major barrier for cost-effective fer-mentative production of butanol. This can be seen from thefact that C. acetobutylicum fermentations rarely produce bu-tanol higher than 13 g/L, a level that is inhibitory for the growthof C. acetobutylicum and is generally considered as the toxiclimit.8 Butanol toxicity in C. acetobutylicum is quite severe, andthis has been attributed to the chaotropic effect on cellmembrane9,10 and the inhibition effects on nutrient transport,glucose uptake, and membrane-bound ATPase activity.9 Mod-

* To whom correspondence should be addressed. Yin Li, Institute ofMicrobiology, Chinese Academy of Sciences, No.1 West Beichen Road,Chaoyang District, Beijing 100101, China. E-mail: yli@im.ac.cn. Tel: +86-10-64807485. Fax: +86-10-64807485.

† These authors contributed equally to this work.‡ Institute of Microbiology, Chinese Academy of Sciences.§ Graduate School of Chinese Academy of Sciences.| State Key Laboratory of Microbial Resources, Institute of Microbiology,

Chinese Academy of Sciences.⊥ University of California.

3046 Journal of Proteome Research 2010, 9, 3046–3061 10.1021/pr9012078 2010 American Chemical SocietyPublished on Web 04/29/2010

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erate increases in butanol titers have been shown to elicit aresponse similar to a heat shock.11 Butanol-stressed exponen-tially growing C. acetobutylicum resulted in upregulation ofnumerous chaperone genes (dnaK, groES, groEL, hsp90, hsp18,clpC, and htrA), solventogenic genes, and glycerol metabolismgenes glpA and glpF.12 Butanol-tolerant strains have beendeveloped from C. acetobutylicum by using classic chemicalmutagenesis, continuous culture, serial enrichment procedures,and targeted genetic/metabolic engineering.13–18 The improvedbutanol tolerance has different impacts on butanol production(decrease,14,18 minor improvement,13,19 or significant (>20%)improvement),15–17 suggesting the relationship between bu-tanol tolerance and butanol production is rather complex andremains largely unknown.

The availability of the complete genome sequence of C.acetobutylicum5 enables the genome-wide investigation of themechanism for butanol tolerance. Genome-wide transcriptomestudies on C. acetobutylicum were performed.12,20,21 Charac-terization of the C. acetobutylicum Spo0A mutant SKO1 by DNAmicroarray analysis revealed that Spo0A inactivation triggereddown-regulation of solventogenic, sporulation, and carbohy-drate metabolism genes and up-regulation of flagellar andchemotaxis genes.22 Transcriptome analysis of C. acetobutylicummutant 824 (pGROE1) with overexpressed groESL revealed anincreased expression of motility and chemotaxis genes.15

Proteomics is a powerful tool to understand the cellular statusat the protein level, which cannot be deciphered from eithergenome or transcriptome analysis. Proteomics approaches areincreasingly employed to identify proteins that can be used astargets for metabolic engineering.23–25 Since two-dimensionalelectrophoresis (2-DE) was introduced as a tool to separatecomplex mixtures of cellular proteins,26 a large number ofprokaryotic proteomes have been studied.27–29 Studies on C.acetobutylicum proteomics are underway. A proteomic analysisof the C. acetobutylicum during the transition from acidogenesisto solventogenesis discovered some changes in the proteinpattern.6 A comparative proteomic analysis between C. aceto-butylicum wild type strain ATCC 824 and its mutant 824(pM-SPOA) discovered that Spo0A overexpression affected theabundance of proteins involved in glycolysis, translation, heatshock stress response, and energy production.7

The aim of this study was to establish a comprehensivecytoplasmic proteome reference map for C. acetobutylicum sothat the reference map can be used as a fundamental tool forcomparative proteomics study to further increase our knowl-edge on the physiology of this species. To illustrate thesignificance of this reference map, we carried out a comparativeproteomic analysis between two strains, C. acetobutylicum wildtype strain DSM 1731 (butanol tolerance 13 g/L) and its mutantRh8 (butanol tolerance 19 g/L), with the aim to obtainfundamental data for understanding the biological mechanismon butanol tolerance and/or butanol yield.

Experimental Section

Bacteria and Culture Conditions. C. acetobutylicum DSM1731, obtained from the German Collection of Microorganismsand Cell Cultures (DSMZ, Braunschweig, Germany), was usedfor generating the proteome reference map. Mutant Rh8 is agenome-shuffled strain derived from strain DSM 1731 and itsbutanol-tolerant mutants. Cells from a single colony were usedto inoculate liquid reinforced clostridial medium (RCM),30

which were then heat shocked at 75 °C for 10 min beforegrowing at 37 °C. When the OD600 reached 1.0, the culture was

stored at 30 °C for one week to produce spores.31 Spore countswere measured as following: 100 µL seed cultures were mixedwith 900 µL deionized distilled water. Disposable micropipetswere used to stir the mixture and drops of the suspensions weretransferred to a hemacytometer slide. An estimate of the totalnumber of spores produced in each chamber was derivedarithmetically from the hemacytometer counts.32 Precultures(10 mL) of strains DSM 1731 and Rh8 were inoculated to about106-107 spores and pasteurized for 10 min at 75 °C beforeincubation, then cultured in a 250 mL bottle containing 100mL of clostridial growth medium (CGM)33 at 37 °C for 12 h. Inall experiments, cell growth was monitored by measuring theabsorbance of the culture broth at 600 nm (OD600) with a modelDU series 800 spectrophotometer (Beckman, Fullerton, CA). Forproteomic analysis, cells were harvested at exponential growthphase at OD600 of 2.0, which is corresponding to 6.5 × 108colony forming units/mL.

Generation of Mutant Rh8. Genome shuffling34 was usedto generate C. acetobutylicum mutant Rh8, which is moretolerant to butanol. C. acetobutylicum DSM 1731 was culturedin reinforced clostridial medium (RCM)30 at 37 °C for 12 h. Cellswere harvested at acid-production phase (OD600 ) 2.0) andresuspended in 0.1 M potassium phosphate buffer (pH 7.0)containing 1% (v/v) diethyl sulfate (DES). The mixture wasincubated at 37 °C for 15 min, and the cells were washed twicewith Trypticase-Glucose-Yeast extract medium (TGY)35 andthen resuspended in RCM and grown at 37 °C for 48 h. Thecultures were plated on RCM agar (RCM + 2% agar) containingvarious amounts of butanol and incubated at 37 °C for 48 h.Colonies grown on RCM agar containing 18 g/L butanol wereselected. Four stable mutants which can tolerate 18 g/L butanolwere subjected to protoplast fusion. Each mutant was grownin clostridial basal medium (CBM)36 containing 0.5% (w/v)glycine at 37 °C for 24 h. Cells were harvested by centrifugationat 3000× g for 10 min, washed twice with isotonic buffer (CBMcontaining 0.5 M sucrose), and treated with isotonic buffercontaining 1 mg/mL lysozyme at 37 °C for 20 min. Protoplastswere harvested by centrifugation at 1000× g for 15 min andgently washed with isotonic buffer. Protoplasts from differentmutants were mixed and divided equally into two parts. Onepart was inactivated with UV for 20 min, and the other partwas inactivated by heating at 80 °C for 30 min. Both inactivatedprotoplasts preparations were mixed and fused by suspensionin 5 mL isotonic buffer containing 40% PEG 6000 at roomtemperature for 5 min. Dilutions of the fused protoplasts wereplated onto a regeneration medium37 and incubated at 37 °Cfor 48 h. Colonies grown on the regeneration medium wereselected to test their tolerance to butanol and the butanolproduction capability. A progeny cell culture that can grow inthe medium containing 19 g/L butanol was obtained.

Fermentation Experiments. Batch fermentation experimentsof C. acetobutylicum DSM 1731 and its mutant Rh8 were carriedout in BioFlo 110 fermentors (New Brunswick Scientific, Edison,NJ) containing 4.0 L (working volumes) of CGM,33 accordingto the cultivation method described in the literature38 withslight modification. Briefly, 200 mL seed cultures were inocu-lated into a fermentor containing 4 L CGM. The initial pH ofthe fermentation was 6.5, and the pH was allowed to drop to5.0 as the culture progressed. Subsequently, the pH wasautomatically maintained at or above 5.0 by adding 6 Mammonium hydroxide. The concentrations of the main me-tabolites in the cell-free fermentation broth (acetate, butyrate,acetone, butanol, ethanol, and glucose) were determined using

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an Agilent 1200 high performance liquid chromatography(Agilent Technologies, Santa Clara, CA). A Bio-Rad AminexHPX-87H ion exchange column (7.8 by 300 mm) (Bio-RadLaboratories, Inc., Richmond, CA) was used with a mobilephase of 0.05 mM sulfuric acid at 15 °C with a flow rate of 0.50mL/min. A refractive index (RI) detector (Agilent) was used at30 °C for signal detection.

Butanol Challenge Experiments. Strain DSM 1731 and itsmutant Rh8 were grown in 500 mL flasks containing 400 mLCGM at 37 °C statically. When OD600 of 1.0 ( 0.05 was achieved(approximately 12 h), each culture was split into four 100 mLaliquots and challenged with 0, 6, 12, and 18 g/L butanol. Thegrowth of these two strains in the presence of differentconcentrations of butanol was further monitored.

Sample Preparation. Cells from exponential growth phase(OD600 ) 2.0) were regarded as acidogenic cells, and from OD600) 5.0 were regarded as solventogenic cells. Cells were harvestedby centrifugation at 10 000× g at 4 °C for 10 min and washedthree times with 45 mL of ice-cold TE buffer (10 mM Tris-HCl,5 mM EDTA, pH 7.5). The resulting C. acetobutylicum DSM1731 and Rh8 cell pellets (about 0.80 g wet weight) wereresuspended in 10 mL of lysis buffer (8 M urea, 2 M thiourea,4% CHAPS (w/v), 50 mM DTT, and 10 mM PMSF) containinga complete protease inhibitors cocktail (Roche diagnostics,Mannheim, Germany). The cells were sonicated on ice for 15min using a Sonifier S-450D (Branson Ultrasonics Corp.,Danbury, CT) with the following conditions: 5 s of sonicationwith a 5-s interval, set at 50% duty cycle. After adding 10 µg/mL nuclease mix (GE Healthcare, Uppsala, Sweden), the celllysate was incubated at ambient temperature for 30-45 minto degrade nucleic acids. The resulting lysate was collected andcentrifuged at 150 000× g in a 90 Ti rotor (Beckman, Fullerton,CA) at 4 °C for 45 min. The supernatant was diluted with threevolume of ice cold acetone. After mixing and incubation at -20°C for 16 h, proteins were sedimented by centrifugation at15 000× g at 4 °C for 30 min. The protein precipitants weresolubilized in sample lysis buffer (8 M urea, 2 M thiourea, 4%CHAPS, 0.5% Pharmalyte pH 3-10). Protein concentration wasmeasured by using the 2-D Quant Kit (GE Healthcare, Uppsala,Sweden), and 1 mg aliquots were stored at -80 °C.

Two-Dimensional Polyacrylamide Gel Electrophoresis. Theextracted proteins from both C. acetobutylicum strain DSM1731 and Rh8 were subjected to 2-DE, respectively. IEF wasperformed on Ettan IPGphor 3 system (GE Healthcare, Uppsala,Sweden) using IPG strips (GE Healthcare). For IPG strips of pH4-7, approximately 1 mg of protein per sample in rehydrationbuffer (8 M urea, 2 M thiourea, 4% CHAPS, 0.5% PharmalytepH 4-7, and 0.001% bromophenol blue) was run using the in-gel sample rehydration technique according to the manufac-turer’s instructions for IEF. IEF was performed using thefollowing voltage program: 30 V constant for 12 h, gradient to200 V for 4 h, gradient to 1000 V within 4 h, gradient to 10 000V within 4 h then 10 000 V for 4 h for a total of 65 000 V ·h. ForIPG strips of pH 6-11, approximately 1 mg protein per sample(100 µL) was loaded on a previously rehydrated strip (rehy-drated for 12 h) by anodic cup loading as recommended bythe manufacturer. IEF was performed using the followingvoltage program slightly modified from previously reported:39

150 V constant for 4 h, gradient to 300 V within 2 h, gradientto 600 V within 2 h, gradient to 8000 V within 30 min and then8000 V until a total of 32 000 V ·h had been achieved. Thetemperature was maintained at 20 °C for IEF.

After completion of the first-dimension IEF, each strip wasequilibrated in 10 mL equilibration buffer 1 (6 M urea, 50 mMDTT, 30% glycerol, 50 mM Tris-HCl, pH 8.8) for 15 min andthen equilibrated in 10 mL equilibration buffer 2 (6 M urea,100 mM iodoacetamide, 30% glycerol, 50 mM Tris-HCl, pH 8.8)for another 15 min. Equilibrated IPG strips were subsequentlyplaced on the top of 12.5% SDS-PAGE gels. A denaturingsolution (0.7% agarose, 0.1% SDS, 192 mM glycine, 25 mM Tris-HCl (pH 8.8), 0.001% bromophenol blue) was loaded onto thegel strips. After agarose solidification, electrophoresis wasperformed in a buffer (pH 8.3) containing 0.3% Tris, 1.44%glycine, and 0.1% SDS, at 16 °C for 1 h at 1 W/gel, followed by5-6 h at 10 W/gel until the bromophenol blue reached thebottom. Six gels were run in parallel on Ettan DALTsix elec-trophoresis system (GE Healthcare). For each condition, 2-DEexperiments were carried out in triplicate. The protein spotswere visualized by Coomassie blue G-250 (Amresco, Solon, OH)staining as previously described.40

2-DE Gel Image Analysis. The gels were scanned at 300 dpiresolution using ImageScannerIII (GE Healthcare). Comparativeanalysis of the protein spots was performed using Image Master6.0 2-D platinum software (GE Healthcare) as previouslydescribed.41 All images were submitted to automatic spotdetection according to the manufacturer’s recommendations.The spots were checked manually to eliminate any possibleartifacts including background noise and streaks. To obviatebatch-to-batch variance, spots that were consistently reproduc-ible in all gel images, including both the biological andtechnical replicates, were chosen for subsequent analyses. Allimages were aligned and matched by using the common spotspresent in all images as landmarks, to detect potential differ-entially expressed proteins. Spot normalization was performedusing relative volumes (%Vol) to quantify and compare the gelspots as described previously,41 with the aim to make the dataindependent of the experimental variations between gels. Onlyprotein spots showing reproducible changes in protein abun-dance, by multiple experiments (at least three biologicalrepetitions and two technical replicates), were considered asbiomarkers associated with wild type strain and mutant.Statistical analysis was performed using Students t-test. P < 0.05was considered significant.

Protein Expression Profile Analysis. Hierarchical clusteringanalysis was used to group proteins exhibiting similar expres-sion profiles. The relative volumes (%Vol) of each protein spot,obtained from the Image Master 6.0 2-D platinum software (GEHealthcare), were used for hierarchical cluster analysis. Theprotein profile normalization was managed as described previ-ously.42 The differentially expressed proteins were grouped onthe basis of similarity in expression profile by using KMCsupport (TIGR MeV, version 4.5.1).43

In-Gel-Digestion. All protein spots were excised and submit-ted to in-gel-digestion as previously described41 with slightmodifications. Briefly, the Coomassie blue-stained protein spotswere manually excised using a spot picker (The Gel Company,San Francisco, CA). The spots were transferred to Eppendorftubes, sealed, and stored at -80 °C until further processing.One-hundred microliters of 50% ACN solution containing 50mM ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO)were added to each tube, and the mixtures were incubated withoccasional vortexing for 30 min. This process was repeated untilall gel spots were completely destained. The spots were thendehydrated with 100 µL of ACN at room temperature for 15min. After ACN removal, the gel spots were dried under vacuum

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and rehydrated in 20 µL of proteomics sequencing grade trypsin(10 ng/µL; Cat.: T6567, Sigma-Aldrich, St. Louis, MO), in whichlysine residues have been reductively methylated, leading toresistance to autolysis. After rehydration at 4 °C for 45 min,excess trypsin solution was removed, and 10 µL of 50 mMammonium bicarbonate was added followed by incubation at37 °C for 16 h. Peptides were extracted twice by the additionof 30 µL of 5% formic acid (v/v)/50% ACN (v/v) solution andvortexing for 30 min. The peptide samples were concentratedby using Speed Vac (Thermo Savant, Waltham, MA) to ap-proximately 10 µL and stored at -20 °C for mass spectrometricanalysis.

MALDI-TOF MS and MS/MS Analysis. A 0.4 µL aliquot ofthe concentrated tryptic peptide mixture in 0.1% TFA wasmixed with 0.4 µL of CHCA matrix solution (5 mg/mL CHCAin 50% ACN/0.1% TFA) and spotted onto a freshly cleanedtarget plate. After air drying, the crystallized spots wereanalyzed on the Applied Biosystems 4700 Proteomics AnalyzerMALDI-TOF/TOF (Applied Biosystems, Framingham, MA). MScalibration was automatically performed by a peptide standardKit (Applied Biosystems) containing des-Arg1-bradykinin (m/z904), Angiotensin I (m/z 1296.6851), Glu1-fibrinopeptide B(m/z 1570.6774), ACTH (1-17, m/z 2903.0867), ACTH (18-39,m/z 2465.1989), and ACTH (7-38, m/z 3657.9294) and MS/MS calibration was performed by the MS/MS fragment peaksof Glu1-fibrinopeptide B. All MS mass spectra were recordedin the reflector positive mode using a laser operated at a 200Hz repetition rate with wavelength of 355 nm. The acceleratedvoltage was operated at 2 kV. The MS/MS mass spectra wereacquired by the data dependent acquisition method with the10 strongest precursors selected from one MS scan. All MS andMS/MS spectra were obtained by accumulation of at least 1000and 3000 laser shots, respectively. Neither baseline subtractionnor smoothing was applied to recorded spectra. MS and MS/MS data were analyzed and peak lists were generated usingGPS Explorer 3.5 (Applied Biosystems). MS peaks were selectedbetween 850 to 3700 Da and filtered with a signal-to-noise ratiogreater than 20. A peak intensity filter was used with no morethan 50 peaks per 200 Da. MS/MS peaks were selected basedon a signal-to-noise ratio greater than 10 over a mass range of60 to 20 Da below the precursor mass. MS and MS/MS datawere analyzed using MASCOT 2.0 search engine (Matrix Sci-ence, London, U.K.) to search against the C. acetobutylicumprotein sequence database (10 159 sequences; 3 116 366 resi-dues) downloaded from NCBI database on March 20 2008.Searching parameters were as follows: trypsin digestion withone missed cleavage, variable modifications (oxidation ofmethionine and carbamidomethylation of cysteine), and themass tolerance of precursor ion and fragment ion at 0.2 Dafor +1 charged ions. For all proteins successfully identified byPeptide Mass Fingerprint and/or MS/MS, Mascot score greaterthan 53 (the default MASCOT threshold for such searches) wasaccepted as significant (p value

Supporting Information 1) and 458 in the pI range of 6-11(Figure 1B, Table S3 of Supporting Information 1). The proteinidentification success rate is 93.5% and the 1206 identifiedprotein spots represent 564 different C. acetobutylicum proteins.368 identified proteins are in the pI range of 4-7 and 243 inthe pI range of 6-11 with 47 proteins overlapping in the pIrange 6-7. A comprehensive list of all identified proteins,including accession numbers, sequence coverage, MOWSEscores, theoretical pI values and molecular weights are shown

in Tables S2 and S3 of Supporting Information 1. Several mem-brane-associated proteins were identified. These include Na+-ABCtransporter (spot 348b), CAC3551), ABC transporter, ATP-bindingprotein (spot 398a), CAC0147), Oligopeptide ABC transporter,periplasmic substrate-binding component (spot 128a)) and severalsubunits (R, �, γ, δ) of ATPase (spots172 a), 264b), 341b), 369b), 376b)

and 384b)). Except that the detection of ATPase subunits has beenreported previously in C. acetobutylicum,9,47 other membrane-associated proteins were detected for the first time in thecytoplasmic proteome of this species.

Functional Classification of the Identified Proteins of C.acetobutylicum. The 564 identified proteins of C. acetobutyli-cum DSM 1731 were grouped into 20 functional categories onthe basis of the Clusters of Orthologous Groups of proteinsclassification scheme (COG)45 (Figure 2). Most of the identifiedproteins were assigned to COG class J (13.1%), which is involvedin proteins biogenesis (Figure 2). The general function predic-tion only group (COG class X, 12.0%) is the second largestcategory of the identified proteins. The third most abundantlyidentified proteins are those proteins involved in amino acidtransport and metabolism (COG class E, 10.3%). The sugarmetabolism-related proteins, especially those proteins involvedin energy metabolism, constitute the fourth largest proportionof the identified proteins (COG class C, 6.2%).

Based on the KEGG database, the identified proteins wereused to reconstruct the metabolic network of C. acetobutylicumDSM 1731 to evaluate the quality and the coverage of theproteome reference map. The results were summarized in TableS4 of Supporting Information 1. Briefly, 416 proteins out of the564 identified proteins could be assigned to 102 metabolicpathways that can be categorized into 18 categories. More than50% of the proteins involved in major metabolic pathways havebeen identified in the reference proteome map. This includes

Figure 1. (A) Reference map of the cytoplasmic proteins from C.acetobutylicum DSM 1731 within pI ranges of 4-7. Approxi-mately 800 µg of protein were subjected to IEF with an IPG 4-7strip (24 cm separation length) and resolved in the seconddimension using 12.5% SDS-PAGE gels. The separated proteinswere stained with Coomassie blue. The identified proteins arelabeled with spot numbers and listed in Table S2 of SupportingInformation 1. (B) Reference proteome map of the cytoplasmicproteins from C. acetobutylicum DSM 1731 within pI ranges of6-11. Approximately 800 µg of protein were subjected to IEF withan IPG 6-11 strip (18 cm separation length) and resolved in thesecond dimension using 12.5% SDS-PAGE gels. The separatedproteins were stained with Coomassie blue. The identifiedproteins are labeled with spot numbers and listed in Table S3 ofSupporting Information 1.

Figure 2. Representation of the distribution into COG categoriesof the predicted and identified C. acetobutylicum DSM 1731proteins. Grouping of proteins into COGs was carried outaccording to the classification scheme provided by the GenBankdatabase.45

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55.5% in carbohydrate metabolism (98/177, indentified proteins/predicted proteins) and 51.2% in energy metabolism (22/43);57.2% in amino acid metabolism (103/180) and 69.0% inmetabolism of other amino acids (20/29); 62.5% in nucleotidemetabolism (40/64) and 65.6% in xenobiotics metabolism (21/32). The ratio of the identified proteins over predicted proteinsinvolved in lipid metabolism (41.7%; 20/48) and cofactorsmetabolism (29.2%; 26/89) is relatively low. Furthermore, thirty-four identified proteins are hypothetical proteins as per genomeannotation. These include 12 proteins with conserved domainand 22 proteins with unknown functions. The production ofthese hypothetical proteins under standard growth conditionssuggests they might be relevant for the physiology and me-tabolism of C. acetobutylicum.

CAI, GRAVY Value Analyses. The codon adaptation index(CAI) of 3848 ORFs of C. acetobutylicum ATCC 824 wascalculated. The CAI of all genes encoding the proteins identifiedon the pH 4-7 and pH 6-11 gels are listed in Table S2 and S3of Supporting Information 1. Comparison of the CAI distribu-tions of genes encoding for proteins identified on the gelsagainst the CAI of all 3848 ORFs is shown in Figure S2 ofSupporting Information 1. The proteins encoded by genes witha CAI value above 0.6 accounts for 85% of the total predictedproteins and 94% of the 564 identified proteins, respectively.This suggests that proteins encoded by genes with a high CAIwere abundant and easily identified, and most of these proteinsare involved in energy metabolism, protein synthesis, andcellular processes. This observation is similar to the resultspreviously reported in Escherichia coli,48 Lactococcus lactis,23,24

Bacillus subtilis,49 and Bifidobacterium longum.50

The GRAVY index is a global descriptor of hydropathy.Proteins with extended hydrophobic regions, such as mem-brane proteins, are difficult to detect under standard gelconditions. By using CodonW software, the GRAVY index wascalculated for hydrophobicity of all identified and theoreticalproteins.51 Only 48 identified proteins had a GRAVY index valueabove zero with the highest being 0.4233. A comparison of theidentified proteins over all theoretical proteins (Figure S3 ofSupporting Information 1) shows that the hydrophobic proteins(with high GRAVY values) were not present among the identi-fied proteins.

Reconstruction of Metabolic Pathways of C. acetobutylicumDSM 1731. The establishment of the proteome reference mapwith a good coverage allowed us to reconstruct the centralmetabolic pathways of C. acetobutylicum DSM 1731. Pathwaysof glycolysis, pentose phosphate pathway, acid and solventformation pathway, fatty acid biosynthesis, purine metabolism,glutamate metabolism, and biosynthesis of valine, leucine andisoleucine were reconstructed and shown in Figure S4 ofSupporting Information 1.

Sugar Metabolism. Nine enzymes that catalyze the reactionsof glycolysis from glucose to pyruvate were identified by thesystematic mapping approach (Figure S4 of Supporting Infor-mation 1). This includes the glucokinase, glucose-6-phosphateisomerase, 6-phosphofructokinase, fructose-bisphosphate al-dolase, glyceraldehyde 3-phosphate dehydrogenase, 3-phos-phoglycerate kinase, phosphoglycerate mutase, enolase, pyru-vate kinase. The only glycolytic enzyme that was not identifiedis triosephosphate isomerase, possibly due to low productionlevel or other reasons. C. acetobutylicum is capable of ferment-ing a wide variety of sugars (e.g., glucose, galactose, cellobiose,mannose, xylose, and arabinose) to acids and solvents. Inrelation to this, mannose-specific phosphotransferase system

component IIAB and phosphomannomutase that catalyze thefirst two reactions of mannose metabolism were identified. Wealso identified UDP-glucose 4-epimerase that converts UDP-galactose to UDP-glucose. Enzymes involved in the catabolismof cellobiose, xylose, and arabinose were not identified in thereference map. This may be attributed to that these enzymeshave not been induced yet under the conditions that glucosewas used as a sole carbon source. Despite this, key enzymes(transketolase, ribulose-phosphate 3-epimerase, and phospho-ribosylpyrop-hosphate synthetase) involved in the synthesis ofphosphoribosyl pyrophosphate (PRPP), an intermediate innucleotide metabolism and the biosynthesis of the amino acidshistidine and tryptophan, were identified.

Acid and Solvent Formation Pathway. With the exceptionof R-acetolactate decarboxylase [EC:4.1.1.5] and alcohol dehy-drogenase [EC:1.1.1.1], the remaining 15 enzymes involved inacid and solvent formation of C. acetobutylicum were allidentified on the proteome reference map. This includes theacetolactate synthase, pyruvate-formate lyase, acetyl-CoA acetyl-transferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxy-butyryl-CoA dehydratase, butyryl-CoA dehydrogenase, bifunc-tional acetaldehyde-CoA/alcohol dehydrogenase, NADH-dependent butanol dehydrogenase, phosphate butyryltransferase,butyrate kinase, butyrate-acetoacetate CoA-transferase, ac-etoacetate decarboxylase, acetolactate synthase, phosphotrans-acetylase, and acetate kinase. This is the first time that thecomprehensive set of proteins involved in solvent formationwas identified at the proteome level. Acetoin produced by C.acetobutylicum during growth is a fairly low level (∼9 mM);52therefore, the production level of the R-acetolactate decar-boxylase catalyzing the last step of the biosynthesis of acetoinmight be too low to be identified. Ethanol is synthesized withacetaldehyde as a precursor by alcohol dehydrogenase. Thisreaction can also be catalyzed by an isoenzyme: NADH-dependent butanol dehydrogenase A [EC:1.1.1.-], which usesacetaldehyde and butyraldehyde almost equally well.53,54 There-fore, alcohol dehydrogenase [EC:1.1.1.1] might not be neededfor the purpose to produce ethanol under the conditions tested.

Amino Acid Metabolism. In the present reference map, 123proteins (about 22% of all functional proteins identified)involved in amino acid metabolism pathway, urea cycle, andmetabolism of amino groups, were identified. Enzymes in-volved in the biosynthesis of valine, leucine, isoleucine, me-tabolism of tryptophan, taurine and hypotaurine, glutamineand glutamate, arginine and ornithine, were all identified,whereas enzymes involved in the metabolism of other aminoacids metabolism pathway were not identified completely. Thisobservation may be attributed to that the complex growthmedium might contain unbalanced amounts of amino acids,so that only some amino acids are synthesized de novo by C.acetobutylicum. The metabolic pathways of major amino acid(isoleucine, valine, leucine, and glutamate) were reconstructedfrom the mapping data and shown in Figure S4 of SupportingInformation 1.

Nucleotide Metabolism, Fatty Acid Synthesis, and OthersProteins. C. acetobutylicum has all genes required for pyrimi-dine and purine nucleotides biosynthesis from PRPP. Thepresent proteome reference map contains 40 enzymes involvedin the metabolism of purine (24 proteins) and pyrimidine (16proteins) nucleotides. The purine biosynthesis pathway hasbeen reconstructed from the proteomic data (Figure S4 ofSupporting Information 1). Other important metabolic path-ways identified from the proteomic data is lipid synthesis

Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant research articles

Journal of Proteome Research • Vol. 9, No. 6, 2010 3051

(Figure S4 of Supporting Information 1). For fatty acid synthesisfrom acetyl-CoA, both acetyl-CoA carboxylase and fatty acidsynthase were detected. However, the ATP citrate lyase thatgenerates cytoplasmic acetyl-CoA from citrate was not identi-fied. Many proteins involved in DNA and RNA synthesis havealso been identified. Notably, more than half of the predictedtranslation factors, including IF-1, IF-2, IF-3, EF-TS, EF-TU, EF-G, EF-P and RF-1, were identified.

Butanol-Tolerant Mutant Rh8 Exhibits Enhanced ButanolYield. We obtained a mutant Rh8 derived from C. acetobutyli-cum DSM 1731 which can tolerate butanol up to 19 g/L,according to the procedure described in the Materials andMethods. Strains DSM 1731 and Rh8 were subjected to variouslevels of butanol challenge (up to 18 g/L) during midexponen-tial growth (OD600 ) 1.0), and the growth after the addition ofbutanol was further monitored (Figure 3). Strain DSM 1731 wasable to grow in the presence of 5 and 12 g/L butanol; howeverthe growth was severely inhibited compared to that of mutantRh8. The mutant Rh8 grew equally well in the presence of 12and 18 g/L butanol. In contrast to this, the wild type strainDSM 1731 was unable to grow when challenged with 18 g/Lbutanol. This shows clearly that mutant Rh8 has a superiorgrowth performance in the presence of butanol as comparedto the wild type strain DSM 1731. In order to gain more insightsinto the physiology of the mutant Rh8, pH-controlled batchfermentations of the wild type strain DSM 1731 and the mutantRh8 were carried out. Data of all measurements from twobiological replicates were averaged and shown in Figure 4. Themutant Rh8 produced 3.9 g/L acetone and 15.3 g/L butanol,

which increased 18 and 23%, respectively, as compared to thewild type strain DSM 1731 (Figure 4A and B). The peakconcentration of butyrate produced by mutant Rh8 (5 g/L) washigher than that of strain DSM 1731 (3 g/L). Strain DSM 1731started to produce metabolites at 24 h (Figure 4A), which isassociated with the rapid consumption of glucose (Figure 4C).However, mutant Rh8 did not start to produce metabolites until30 h (Figure 4B). Moreover, although it takes longer for mutantRh8 to reach the maximum average butanol productivity thanthat of strain DSM 1731, and the maximum average butanolproductivity of mutant Rh8 was slightly lower (Figure 4F), theaverage butanol yield of mutant Rh8 was much higher thanthat of strain DSM 1731 (Figure 4E). Notably, the specificgrowth rate of strain Rh8 after incubation for 30 h was lowerthan that of DSM 1731 (Figure 4A and B). This might be due tothat Rh8 produced more butyrate (5 g/L) than that by DSM1731 (3 g/L) in such a pH-controlled fermentation, resultingin growth inhibition.

Comparative Proteomic Analysis between C. acetobutyli-cum DSM 1731 and Mutant Rh8. To better understand therelationship between butanol tolerance and butanol yield, weperformed comparative proteomic analysis (pH 4-7 and 6-11)between the wild type strain DSM 1731 and the mutant Rh8using the established proteome reference map. The proteomesof both cells harvested at the acidogenic and solventogenicphases were compared (Table 1, Figure S1, S2, and S3 ofSupporting Information 2). A total of 102 significantly differ-entially expressed proteins were identified. The majority of thedifferentially expressed proteins were involved in the protein

Figure 3. Growth profiles of the wild type strain DSM 1731 (open circles) and the mutant Rh8 (solid circles) challenged with differentconcentration of butanol. (A) 0 g/l butanol; (B) 5 g/l butanol; (C) 12 g/l butanol; (D) 18 g/l butanol.

research articles Mao et al.

3052 Journal of Proteome Research • Vol. 9, No. 6, 2010

http://pubs.acs.org/action/showImage?doi=10.1021/pr9012078&iName=master.img-003.png&w=450&h=336

folding (16 proteins), solvent formation (10), amino acidmetabolism (12), protein synthesis (11), nucleotide metabolism(9) transport (7), and others. Of the 102 proteins, the genescoding for 52 proteins were reported to respond to butanolstress or the transition from acidogenesis to solvento-genesis,6,12,15,20,21,55,56 while the other 50 proteins have not beenpreviously reported to be related to butanol tolerance or thetransition from acidogenesis to solventogenesis (labeled as

asterisk in Figure 5). We compared the profiles of differentiallyexpressed proteins of C. acetobutylicum DSM 1731 and Rh8 withthe profiles of differentially expressed genes of C. acetobutylicumATCC 82456 (Figure S5 of Supporting Information 1). The resultsshowed that only part of the DNA microarray data overlappedwith the differentially expressed protein that we identified.

To explore the difference between the proteomes of strainsDSM 1731 and Rh8 during acidogenesis and solventogenesis,

Figure 4. Time-courses of the batch fermentations (pH > 5.0) of (A) the wild type strain DSM 1731 and (B) the mutant Rh8. Arrowsindicate samples were withdrawn for proteome analysis. (C) Glucose utilization profiles; (D) pH profiles; (E) average butanol yieldprofiles; (F) average butanol productivity profiles. DSM 1731 (open symbols) and Rh8 (solid symbols).

Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant research articles

Journal of Proteome Research • Vol. 9, No. 6, 2010 3053

http://pubs.acs.org/action/showImage?doi=10.1021/pr9012078&iName=master.img-004.png&w=490&h=556

Tab

le1.

Fun

ctio

nal

Cla

ssifi

cati

on

of

Dif

fere

nt

Pro

tein

sin

C.

acet

ob

uty

licu

mD

SM

1731

and

its

Mu

tan

tR

h8

rati

og

(P-v

alu

e)

mat

chid

ap

rote

ind

escr

ipti

on

b

NC

BI

acce

ssio

nn

um

ber

theo

r.M

rc/

pId

gen

elo

cus

gen

esq

uen

ceco

vera

ge(%

)

pep

tid

esm

atch

ed/

sear

ched

e

un

iqu

ep

epti

des

det

ecte

db

yM

S/M

SfM

ASC

OT

sco

re

Rh

8ac

ido

gen

esis

/DSM

1731

acid

oge

nes

is

DSM

1731

/so

lven

toge

nes

is/

DSM

1731

acid

oge

nes

is

Rh

8so

lven

toge

nes

is/

Rh

8ac

ido

gen

esis

Car

bo

hyd

rate

met

abo

lism

1gl

uco

se-6

-ph

osp

hat

eis

om

eras

egi

|150

2571

149

759/

5.37

CA

C26

80p

gi61

111

42.

1(2

.31

×10

-3 )

1.8

(2.7

0×

10-

3 )N

S2

Ph

osp

ho

man

no

mu

tase

gi|1

5025

346

6467

4/5.

59C

AC

2337

4534

/69

-16

91.

4(1

.85

×10

-3 )

NS

2.5

(2.4

0×

10-

4 )3

Tra

nsk

eto

lase

gi|1

5023

846

7266

2/5.

21C

AC

0944

tkt

3720

/33

97N

S-

3.3

(5.1

2×

10-

4 )-

3.8

(2.4

8×

10-

4 )4

Pyr

uva

teki

nas

e(p

ykA

)gi

|150

2338

150

560/

5.74

CA

C05

18p

ykA

606

368

NS

-2.

3(1

.08

×10

-2 )

-3.

2(2

.57

×10

-5 )

5A

ceto

lact

ate

syn

thas

ela

rge

sub

un

itgi

|150

2623

760

075/

5.35

CA

C31

69il

vB46

121

0N

Dh

ND

hN

S6

Pyr

uva

te-f

orm

ate

lyas

egi

|150

2388

784

406/

6.24

CA

C09

80p

flB

556

490

NS

-3.

4(5

.38

×10

-4 )

-12

.3(9

.96

×10

-3 )

7A

cety

l-C

oA

acet

yltr

ansf

eras

e(t

hio

lase

A)

gi|1

5025

920

4144

3/6.

92C

AC

2873

thl

621

220

2.0

(2.4

3×

10-

4 )N

SN

S

8A

ceta

teki

nas

egi

|150

2471

144

313/

6.23

CA

C17

43as

kA60

550

9N

S-

1.7

(2.7

4×

10-

2 )-

2.0

(1.5

9×

10-

3 )9

bif

un

ctio

nal

acet

ald

ehyd

e-C

oA

/al

coh

ol

deh

ydro

gen

ase

gi|1

5004

865

9577

4/8.

44C

AP

0162

adh

e126

23/6

8-

113

1.7

(1.2

1×

10-

2 )5.

7(3

.42

×10

-4 )

3.5

(1.8

7×

10-

2 )

10B

uty

rate

-ace

toac

etat

eC

OA

-tra

nsf

eras

esu

bu

nit

A(C

tfA

)gi

|150

0486

623

797/

8.99

CA

P01

63ct

fA82

458

02.

7(3

.54

×10

-3 )

3.6

(1.8

4×

10-

3 )2.

7(9

.90

×10

-3 )

11B

uty

rate

-ace

toac

etat

eC

OA

-tra

nsf

eras

esu

bu

nit

B(C

tfB

)gi

|150

0486

723

666/

7.79

CA

P01

64ct

fB36

113

22.

7(9

.57

×10

-3 )

15.6

(3.0

6×

10-

5 )7.

7(4

.07

×10

-5 )

12A

ceto

acet

ate

dec

arb

oxy

lase

gi|1

5004

868

2769

0/5.

81C

AP

0165

adc

675

314

2.7

(1.2

8×

10-

3 )2.

5(2

.44

×10

-3 )

NS

13P

ho

sph

ate

bu

tyry

ltra

nsf

eras

egi

|150

2613

932

207/

6.34

CA

C30

76p

tb71

561

2N

S2.

4(2

.77

×10

-4 )

NS

14N

AD

H-d

epen

den

tb

uta

no

ld

ehyd

roge

nas

eB

(BD

HII

)gi

|150

2637

643

259/

5.75

CA

C32

98bd

hB

585

432

4.1

(3.7

6×

10-

4 )N

S-

2.9

(7.6

9×

10-

4 )

15N

AD

H-d

epen

den

tb

uta

no

ld

ehyd

roge

nas

eA

(BD

HI)

gi|1

5026

377

4318

3/5.

81C

AC

3299

bdh

A31

172

3.2

(4.4

8×

10-

3 )N

S-

1.9

(1.3

6×

10-

2 )

16A

con

itas

eA

gi|1

5023

877

6959

0/5.

83C

AC

0971

citB

3316

/33

-97

NS

-2.

1(1

.27

×10

-2 )

-2.

4(7

.75

×10

-4 )

17B

ioti

nca

rbo

xyl

carr

ier

pro

tein

of

acet

yl-C

oA

carb

oxy

lase

gi|1

5026

668

1784

5/4.

50C

AC

3572

accB

746

310

NS

2.1

(8.3

9×

10-

3 )1.

3(4

.90

×10

-3 )

18M

alic

enzy

me

gi|1

5024

543

5945

0/5.

78C

AC

1589

mal

S64

737

2N

S-

2.5

(4.8

7×

10-

2 )-

6.0

(1.0

0×

10-

5 )19

Pyr

uva

te:f

erre

do

xin

oxi

do

red

uct

ase

gi|1

5025

228

1286

00/5

.88

CA

C22

2941

668

7N

S-

2.2

(4.3

3×

10-

3 )N

S20

Tet

rah

ydro

fola

ted

ehyd

roge

nas

e/cy

clo

hyd

rola

segi

|150

2507

130

669/

6.34

CA

C20

83fo

lD46

498

NS

1.5

(3.2

9×

10-

2 )2.

5(3

.17

×10

-4 )

En

ergy

met

abo

lism

21E

xop

oly

ph

osp

hat

ase

gi|1

5025

130

3467

4/5.

42C

AC

2138

754

416

2.3

(1.3

7×

10-

3 )N

S-

2.2

(4.9

0×

10-

2 )22

Fer

red

oxi

n-n

itri

tere

du

ctas

egi

|150

2291

858

881/

6.44

CA

C00

9474

668

9-

1.5

(1.8

7×

10-

2 )-

2.7

(8.5

0×

10-

4 )-

2.7

(6.9

9×

10-

3 )23

Fla

vop

rote

ingi

|150

2393

844

462/

5.23

CA

C10

2761

45/6

9-

171

1.7

(4.7

0×

10-

5 )N

SN

S24

flav

od

oxi

ngi

|150

2547

015

611/

4.80

CA

C24

5264

529

1N

SN

S9.

0(3

.93

×10

-6 )

25R

ibo

flav

insy

nth

ase

bet

a-ch

ain

gi|1

5023

462

1647

6/6.

14C

AC

0593

ribH

663

338

-1.

7(1

.81

×10

-2 )

NS

2.9

(1.0

8×

10-

3 )26

Nif

Uh

om

olo

gue

invo

lved

inF

e-S

clu

ster

form

atio

ngi

|150

2637

015

918/

4.91

CA

C32

9269

320

3N

S2.

8(2

.33

×10

-3 )

NS

Am

ino

acid

and

pro

tein

syn

thes

is-r

elat

edp

rote

ins

27T

hre

on

yl-t

RN

Asy

nth

etas

egi

|150

2537

373

150/

5.68

CA

C23

62th

rS43

121

1-

2.3

(3.1

7×

10-

4 )-

6.0

(5.9

9×

10-

4 )-

4.9

(8.2

0×

10-

5 )28

Pro

lyl-

tRN

Asy

nth

etas

egi

|150

2624

764

096/

5.37

CA

C31

78p

roS

383

107

NS

-2.

5(3

.23

×10

-4 )

-2.

6(4

.79

×10

-4 )

29G

lycy

l-tR

NA

syn

thet

ase

gi|1

5026

266

5334

3/5.

30C

AC

3195

glyS

637

471

NS

-2.

6(4

.12

×10

-4 )

-2.

6(2

.28

×10

-4 )

30G

luta

myl

-tR

NA

syn

thet

ase

gi|1

5023

897

5543

6/5.

41C

AC

0990

gltX

595

416

NS

NS

-2.

4(1

.52

×10

-4 )

31A

spar

tyl-

tRN

Asy

nth

etas

egi

|150

2527

368

200/

5.24

CA

C22

69as

pS

3015

/39

-62

NS

-6.

1(3

.20

×10

-3 )

-5.

8(8

.22

×10

-3 )

32M

eth

ion

yl-t

RN

Asy

nth

etas

egi

|150

2604

773

571/

5.57

CA

C29

91m

etS

6241

/58

-20

3N

SN

S-

2.6

(2.1

1×

10-

3 )33

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l-tR

NA

syn

thet

ase

gi|1

5022

836

4862

2/5.

98C

AC

0017

serS

607

541

NS

-2.

0(2

.54

×10

-2 )

-2.

6(2

.52

×10

-5 )

34A

min

otr

ansf

eras

egi

|150

2283

339

952/

6.23

CA

C00

14se

rC71

662

6N

S-

1.5

(4.4

1×

10-

2 )-

2.6

(8.7

9×

10-

3 )35

D-3

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osp

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ed

ehyd

roge

nas

egi

|150

2283

433

283/

6.17

CA

C00

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1-

160

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0(1

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elat

edto

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Hd

om

ain

of

Spo

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Par

A/P

arB

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Bfa

mily

gi|1

5022

835

4769

3/5.

78C

AC

0016

433

172

NS

-2.

6(5

.57

×10

-4 )

-1.

9(3

.20

×10

-2 )

37D

ihyd

roxy

-aci

dd

ehyd

rata

segi

|150

2623

858

329/

6.02

CA

C31

70il

vD40

382

NS

NS

-2.

4(1

.01

×10

-2 )

38Is

op

rop

ylm

alat

ed

ehyd

roge

nas

egi

|150

2623

938

955/

5.01

CA

C31

71le

uB

456

194

NS

NS

2.0

(1.5

8×

10-

2 )39

3-is

op

rop

ylm

alat

ed

ehyd

rata

se,

smal

lsu

bu

nit

gi|1

5026

240

1801

3/5.

74C

AC

3172

leu

D32

212

2N

SN

S2.

5(2

.96

×10

-3 )

40U

DP

-N-a

cety

lmu

ram

yltr

ipep

tid

esy

nth

ase,

MU

RE

gi|1

5025

861

5381

1/5.

52C

AC

2819

mu

rE67

439

9N

S-

8.4

(3.7

4×

10-

3 )N

S

41G

luta

min

esy

nth

etas

ety

pe

III

gi|1

5025

687

7669

4/5.

56C

AC

2658

gln

A36

21/3

4-

113

NS

-4.

8(2

.65

×10

-4 )

-6.

9(3

.29

×10

-2 )

research articles Mao et al.

3054 Journal of Proteome Research • Vol. 9, No. 6, 2010

Tab

le1.

Co

nti

nu

ed

rati

og

(P-v

alu

e)

mat

chid

ap

rote

ind

escr

ipti

on

b

NC

BI

acce

ssio

nn

um

ber

theo

r.M

rc/

pId

gen

elo

cus

gen

esq

uen

ceco

vera

ge(%

)

pep

tid

esm

atch

ed/

sear

ched

e

un

iqu

ep

epti

des

det

ecte

db

yM

S/M

SfM

ASC

OT

sco

re

Rh

8ac

ido

gen

esis

/DSM

1731

acid

oge

nes

is

DSM

1731

/so

lven

toge

nes

is/

DSM

1731

acid

oge

nes

is

Rh

8so

lven

toge

nes

is/

Rh

8ac

ido

gen

esis

42G

lu-t

RN

AG

lnam

ido

tran

sfer

ase

sub

un

itA

gi|1

5025

700

5317

5/5.

47C

AC

2670

446

355

NS

-1.

5(4

.97

×10

-3 )

-2.

7(1

.08

×10

-4 )

43G

lyci

ne

hyd

roxy

met

hyl

tran

sfer

ase

gi|1

5025

267

4558

3/6.

16C

AC

2264

glyA

595

373

NS

-2.

3(3

.35

×10

-3 )

NS

44D

AH

Psy

nth

ase

rela

ted

pro

tein

gi|1

5023

790

3719

1/6.

16C

AC

0892

7335

/70

-12

4N

S-

2.6

(1.9

1×

10-

3 )-

2.8

(1.4

7×

10-

3 )45

Cys

tein

esy

nth

ase/

cyst

ath

ion

ine

bet

a-sy

nth

ase,

Cys

Kgi

|150

2523

532

918/

5.54

CA

C22

35cy

sK70

34/5

9-

158

NS

1.9

(3.6

0×

10-

3 )2.

3(1

.66

×10

-4 )

46B

ran

ched

-ch

ain

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ino

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dtr

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min

ase

gi|1

5024

425

3748

8/5.

94C

AC

1479

ilvE

5924

/68

63N

SN

S-

2.1

(6.0

0×

10-

4 )

47U

DP

-N-a

cety

lglu

cosa

min

ep

yro

ph

osp

ho

ryla

segi

|150

2629

549

661/

6.01

CA

C32

22gc

aD47

522

6N

S-

1.4

(4.8

8×

10-

2 )-

2.0

(1.6

3×

10-

3 )

48R

ibo

som

alp

rote

inL4

gi|1

5026

198

2285

1/9.

78C

AC

3132

rplD

684

442

NS

NS

-4.

7(9

.52

×10

-3 )

49Sp

oO

Jre

gula

tor,

soj/

par

afa

mily

gi|1

5004

880

2861

8/5.

42C

AP

0177

434

264

NS

2.3

(2.7

7×

10-

4 )2.

0(1

.50

×10

-2 )

50F

erri

cu

pta

kere

gula

tio

np

rote

ingi

|150

2464

517

711/

5.59

CA

C16

8283

519

7N

SN

S2.

4(6

.94

×10

-3 )

51R

ibo

som

e-as

soci

ated

pro

tein

Y(P

Srp

-1)

gi|1

5025

892

2028

1/5.

59C

AC

2847

596

339

NS

1.4

(8.3

8×

10-

3 )3.

2(1

.03

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Mem

bra

ne

tran

spo

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Iro

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late

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BC

tran

spo

rter

AT

Pas

esu

bu

nit

(Su

fC)

gi|1

5026

366

2742

2/5.

83C

AC

3288

815

503

1.6

(2.8

9×

10-

3 )1.

6(2

.42

×10

-2 )

NS

53A

BC

-typ

ep

ola

ram

ino

acid

tran

spo

rtsy

stem

,A

TP

ase

com

po

nen

tgi

|150

2377

527

293/

6.93

CA

C08

7945

379

NS

2.4

(8.2

5×

10-

3 )N

S

54F

oF

1-ty

pe

AT

Psy

nth

ase

alp

ha

sub

un

itgi

|150

2591

355

198/

5.21

CA

C28

67at

pA

476

632

NS

-2.

4(1

.62

×10

-2 )

-4.

5(1

.74

×10

-6 )

55F

oF

1-ty

pe

AT

Psy

nth

ase

bet

asu

bu

nit

gi|1

5025

911

5106

2/4.

87C

AC

2865

atp

D77

779

5N

S-

1.7

(2.0

7×

10-

2 )-

2.4

(7.7

1×

10-

4 )

56F

oF

1-ty

pe

AT

Psy

nth

ase

gam

ma

sub

un

itgi

|150

2591

231

175/

9.19

CA

C28

66at

pG

472

197

NS

3.0

(2.9

8×

10-

3 )N

S

57A

BC

tran

spo

rter

,A

TP

-bin

din

gp

rote

ingi

|150

2389

135

696/

6.97

CA

C09

8452

110

9N

S5.

1(5

.33

×10

-4 )

NS

58N

a+-A

BC

tran

spo

rter

(AT

P-b

ind

ing

pro

tein

),N

AT

Agi

|150

2664

626

659/

6.56

CA

C35

51n

atA

492

205

2.2

(2.9

0×

10-

3 )-

2.0

(5.7

9×

10-

3 )N

S

Nu

cleo

tid

em

etab

olis

m59

GT

Pas

e,su

lfat

ead

enyl

ate

tran

sfer

ase

sub

un

it1

gi|1

5022

935

5891

9/5.

74C

AC

0110

cysN

4326

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-90

2.1

(1.4

3×

10-

2 )7.

1(2

.92

×10

-4 )

3.4

(1.6

6×

10-

3 )

60P

oly

rib

on

ucl

eoti

de

nu

cleo

tid

yltr

ansf

eras

egi

|150

2478

177

940/

5.28

CA

C18

08p

np

A30

223

9-

1.6

(3.0

9×

10-

3 )-

6.6

(1.0

7×

10-

4 )-

5.2

(1.8

8×

10-

3 )

61C

TP

syn

thas

egi

|150

2593

960

042/

5.42

CA

C28

92ct

rA42

22/3

0-

151

NS

-2.

4(7

.29

×10

-4 )

-4.

1(1

.11

×10

-3 )

62O

rota

tep

ho

sph

ori

bo

sylt

ran

sfer

ase

gi|1

5022

847

2543

9/5.

68C

AC

0027

pyr

E61

314

2N

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4(7

.77

×10

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1.4

(6.1

7×

10-

3 )63

Ad

enin

ep

ho

sph

ori

bo

sylt

ran

sfer

ase

gi|1

5025

279

1895

7/4.

94C

AC

2275

apt

885

380

NS

1.6

(3.2

6×

10-

3 )2.

0(9

.53

×10

-3 )

64N

ud

ix(M

utT

)fa

mil

yh

ydro

lase

gi|1

5024

831

1985

5/5.

76C

AC

1854

502

79N

SN

S2.

4(6

.38

×10

-3 )

65U

rid

ylat

eki

nas

egi

|150

2476

125

837/

5.82

CA

C17

89sm

bA61

539

3N

S3.

0(2

.51

×10

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3.4

(4.5

8×

10-

3 )66

DN

Ap

oly

mer

ase

III

bet

asu

bu

nit

gi|1

5022

819

4108

8/4.

83C

AC

0002

dn

aN62

630

6N

SN

S2.

1(1

.24

×10

-2 )

67R

EC

Are

com

bin

ase,

AT

Pas

egi

|150

2478

938

179/

6.88

CA

C18

15re

cA45

111

3N

S6.

7(1

.89

×10

-4 )

NS

Ch

aper

on

ep

rote

ins

68M

ole

cula

rch

aper

on

e,H

SP90

fam

ily

gi|1

5026

394

7236

1/5.

09C

AC

3315

htp

G47

227

72.

2(1

.08

×10

-3 )

1.3

(2.6

7×

10-

3 )N

S69

Mo

lecu

lar

chap

ero

ne

(sm

all

hea

tsh

ock

pro

tein

),H

SP18

gi|1

5026

820

1773

4/5.

27C

AC

3714

hsp

1869

446

61.

8(7

.86

×10

-5 )

2.7

(9.5

4×

10-

4 )2.

4(1

.25

×10

-6 )

70C

hap

ero

nin

Gro

EL,

HSP

60fa

mil

ygi

|150

2573

658

038/

4.89

CA

C27

03gr

oEL

4930

/67

-70

1.7

(1.2

3×

10-

3 )1.

7(7

.35

×10

-3 )

NS

71M

ole

cula

rch

aper

on

eG

rpE

gi|1

5024

209

2270

5/4.

50C

AC

1281

grp

E58

115

41.

5(6

.00

×10

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1.4

(9.0

4×

10-

3 )2.

7(6

.00

×10

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72M

ole

cula

rch

aper

on

eD

naK

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SP70

fam

ily

gi|1

5024

210

6560

9/4.

80C

AC

1282

dn

aK48

549

21.

4(3

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×10

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NS

NS

73A

TP

ase

wit

hch

aper

on

eac

tivi

tycl

pC

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oA

TP

-bin

din

gd

om

ain

gi|1

5026

259

9197

8/5.

94C

AC

3189

clp

C22

334

01.

5(1

.77

×10

-2 )

NS

6.9

(1.2

2×

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4 )

74A

rgin

ine

kin

ase

rela

ted

enzy

me,

YA

CI

B.s

ub

tili

so

rth

olo

ggi

|150

2626

038

785/

5.42

CA

C31

90ya

cI58

554

4N

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9(1

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×10

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1.5

(1.6

3×

10-

3 )

75H

trA

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rin

ep

rote

ase

(wit

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DZ

do

mai

n)

gi|1

5025

449

4555

6/6.

02C

AC

2433

htr

A54

116

42.

3(9

.10

×10

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NS

NS

76C

o-c

hap

ero

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Gro

ES,

HSP

10fa

mily

gi|1

5025

737

1042

0/5.

06C

AC

2704

groE

S86

538

3N

S2.

2(3

.65

×10

-3 )

2.3

(3.9

9×

10-

3 )77

Ru

bre

ryth

rin

gi|1

5026

696

2009

4/5.

50C

AC

3598

765

400

NS

NS

1.5

(3.0

2×

10-

2 )

Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant research articles

Journal of Proteome Research • Vol. 9, No. 6, 2010 3055

Tab

le1.

Co

nti

nu

ed

rati

og

(P-v

alu

e)

mat

chid

ap

rote

ind

escr

ipti

on

b

NC

BI

acce

ssio

nn

um

ber

theo

r.M

rc/

pId

gen

elo

cus

gen

esq

uen

ceco

vera

ge(%

)

pep

tid

esm

atch

ed/

sear

ched

e

un

iqu

ep

epti

des

det

ecte

db

yM

S/M

SfM

ASC

OT

sco

re

Rh

8ac

ido

gen

esis

/DSM

1731

acid

oge

nes

is

DSM

1731

/so

lven

toge

nes

is/

DSM

1731

acid

oge

nes

is

Rh

8so

lven

toge

nes

is/

Rh

8ac

ido

gen

esis

78P

rote

ase

sub

un

its

of

AT

P-d

epen

den

tp

rote

ase,

Clp

Pgi

|150

2566

721

410/

5.03

CA

C26

40cl

pP

323

101

NS

NS

2.6

(5.3

7×

10-

3 )

79P

hag

esh

ock

pro

tein

Agi

|150

2315

624

964/

5.35

CA

C03

1372

335

6N

SN

S1.

4(6

.55

×10

-3 )

80M

eth

ion

ine

amin

op

epti

das

egi

|603

9122

727

284/

5.14

CA

C31

11m

ap74

542

7N

S1.

3(1

.10

×10

-2 )

1.6

(1.1

4×

10-

4 )81

Elo

nga

tio

nF

acto

rT

u(E

f-T

u)

gi|1

5026

203

4342

5/5.

04C

AC

3136

tufA

363

135

NS

2.3

(4.1

8×

10-

4 )2.

2(8

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82T

ran

scri

pti

on

elo

nga

tio

nfa

cto

r,gr

eAgi

|150

2626

817

701/

4.70

CA

C31

98gr

eA82

531

2N

SN

S2.

6(6

.91

×10

-4 )

83N

-Ter

min

alfr

agm

ent

of

elo

nga

tio

nfa

cto

rT

sgi

|150

2422

621

921/

4.92

CA

C12

9726

210

6N

SN

S3.

6(1

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Oth

ers

84P

oss

ible

ho

ok-

asso

ciat

edp

rote

in,

flag

ellin

fam

ilygi

|150

2519

929

503/

5.78

CA

C22

03h

ag58

346

7-

12.3

(4.4

7×

10-

5 )-

3.5

(3.0

4×

10-

4 )1.

7(1

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85C

ell

div

isio

nG

TP

ase

Fts

Zgi

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2465

739

365/

4.87

CA

C16

93ft

sZ77

666

2N

S-

4.3

(2.9

7×

10-

4 )N

S86

Cel

ld

ivis

ion

pro

tein

Div

IVA

gi|1

5025

108

2405

6/4.

57C

AC

2118

706

296

NS

NS

2.0

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7×

10-

2 )87

ER

AG

TP

ase

gi|1

5024

224

3383

3/9.

05C

AC

1295

361

60N

SN

S4.

4(3

.69

×10

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88C

hem

ota

xis

pro

tein

Ch

eBgi

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2522

038

121/

8.86

CA

C22

22ch

eB46

317

6N

SN

S2.

7(1

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89P

red

icte

dp

ho

sph

ate-

uti

lizi

ng

enzy

me

invo

lved

inp

yrid

oxi

ne/

pu

rin

e/h

isti

din

eb

iosy

nth

esis

gi|1

5023

463

3181

2/5.

50C

AC

0594

pd

xY73

563

3-

2.7

(6.6

8×

10-

5 )-

4.9

(2.1

5×

10-

3 )1.

2(4

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90D

NA

gyra

se(t

op

ois

om

eras

eII

)B

sub

un

itgi

|150

2282

371

570/

5.80

CA

C00

06gy

rB24

15/2

0-

99-

1.6

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1×

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3 )-

2.6

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3 )N

S

91F

usi

on

of

Uro

po

rph

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oge

n-I

IIm

eth

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ere

late

dp

rote

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dM

AZ

Gfa

mil

yp

rote

in,

YA

BN

B.s

ub

tilis

gi|1

5026

284

5573

1/4.

92C

AC

3212

483

110

NS

5.8

(3.7

1×

10-

4 )N

S

92N

itro

red

uct

ase

fam

ilyp

rote

ingi

|150

2450

219

323/

6.64

CA

C15

5152

114

44.

7(4

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-4 )

NS

NS

93P

rote

inco

nta

inin

gce

llad

hes

ion

do

mai

ngi

|150

2615

054

539/

4.76

CA

C30

8625

110

6N

Dh

ND

hN

S

94N

itro

red

uct

ase

fam

ilyp

rote

ingi

|150

2639

322

826/

5.70

CA

C33

1467

421

7N

SN

S2.

7(3

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×10

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95P

uta

tive

met

hyl

tran

sfer

ase

gi|1

5023

435

2420

6/5.

50C

AC

0567

364

243

NS

NS

3.9

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7×

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6 )96

PP

-lo

op

sup

erfa

mily

AT

Pas

e,co

nfe

rsal

um

inu

mre

sist

ance

gi|1

5026

727

2549

4/8.

04C

AC

3627

313

61N

S2.

9(1

.46

×10

-3 )

NS

97M

ccF

-lik

ep

rote

ingi

|150

2313

534

721/

6.51

CA

C02

9333

211

9N

S-

2.2

(3.7

1×

10-

3 )N

S98

Qu

euin

etR

NA

-rib

osy

ltra

nsf

eras

egi

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2528

743

200/

7.2

CA

C22

82tg

t38

387

NS

3.9

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3 )8.

7(1

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99P

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den

zym

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ith

TIM

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rel

fold

gi|1

5022

975

2459

6/8.

99C

AC

0148

434

257

NS

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5×

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4 )10

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100

hyp

oth

etic

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rote

inC

AC

0488

gi|1

5023

348

1350

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05C

AC

0488

411

111

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3 )N

SN

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1H

ypo

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ical

pro

tein

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F-2

1fa

mil

ygi

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2318

019

463/

5.09

CA

C03

3439

317

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102

Hyp

oth

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AC

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543

165

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Fig

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research articles Mao et al.

3056 Journal of Proteome Research • Vol. 9, No. 6, 2010

the hierarchical clustering analysis and heat map visualizationwere performed with TIGR MeV.43 The hierarchical clusteringanalysis allowed the discrimination of four distinct profiles ofprotein expression (Figure 5). Cluster 1 contained 38 proteinsupregulated in solventogenesis, whereas the protein expressionlevels in mutant Rh8 were generally higher than that of strainDSM 1731 in both acidogenesis and solventogenesis. Themajority of proteins (17/38) grouped in this cluster are involvedin solvent formation, protein folding, cellular regulation, andcell division (Figure 5). Cluster 2 contained 15 proteins thatwere generally stable between acidogenic and solventogenicphases but significantly upregulated in mutant Rh8. Seven outof 15 are involved in solvent formation, protein folding, andtransport. Cluster 3 contained 36 proteins downregulated insolventogenesis, whereas the protein expression levels inmutant Rh8 were generally lower than that of strain DSM 1731in both acidogenesis and solventogenesis. Interestingly, 19 outof 36 are involved in amino acid metabolism and proteinsynthesis. Cluster 4 contained 13 proteins slightly upregulated

in solventogenesis, whereas the protein expression levels ofstrain Rh8 was generally lower than that of DSM 1731 insolventogenesis. Proteins grouped in this cluster were mainlyinvolved in nucleotide metabolism and transport.

Transcriptional Analysis of Selected Genes by Real TimeRT-PCR. To investigate whether the proteins showing alteredlevels on 2-DE are in good accordance with the changes at thetranscriptional level, we selected 11 genes whose encodedproteins were found differentially regulated on 2-DE and themRNA transcript levels were measured by using real time RT-PCR. Interestingly, transcriptional regulation of all selectedgenes showed a positive correlation with the proteomic pat-terns of the identified proteins. Genes corresponding to theproteins that were up-regulated in proteomic studies, such asilvB, ctfA, ctfB, groEL, hsp18, dnaK, natA, bdhB, adhE1, and thl,were also up-regulated at the mRNA level (Figure 6). Hag thatwas down-regulated at the proteome level was also down-regulated at the mRNA level (hag) (Figure 6).

Figure 5. Hierarchical clustering of proteomic data performed using TIGR MeV software. Two phases analysis of differential proteinsaccording to C. acetobutylicum DSM 1731 and Rh8. The relative expression levels of differential expression proteins were hierarchicalclustering analysis according to relative volumes (%Vol) of the protein spots. Sol, solventogenesis; Acid, acidogenesis; 1731, DSM1731. Proteins that have not been previously reported to be related to butanol tolerance or the transition from acidogenesis tosolventogenesis were labeled as asterisk.

Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant research articles

Journal of Proteome Research • Vol. 9, No. 6, 2010 3057

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Discussion

A high resolution 2-DE proteome reference map of C.acetobutylicum is essential for comparative proteomic analysesto investigate the difference among different growth stages, oramong the wild type and the various mutants. Previousproteomic analysis of C. acetobutylicum mainly focused onacidic proteins6,7 here we cover both acidic and basic proteins.We identified 564 proteins account for 14.7% of the predicted3848 ORFs in the genome. This percentage is in a goodaccordance with the recently published proteome referencemaps of other microorganisms, where the percentage of theidentification proteins is usually between 5-21.3% (e.g., 5.9%for Corynebacterium efficiens YS-314,57 4.6% for Corynebacte-rium glutamicum ATCC 14067,58 5.7% for Bacillus anthracisA16R,59 12.8% for Neisseria meningitidis serogroup A,60 and21.3% for Bifidobacterium longum NCC270550). The identifiedproteins were used to reconstruct the metabolic network of C.acetobutylicum DSM 1731 to evaluate the quality and thecoverage of the proteome reference map. More than 50% ofthe proteins involved in major metabolic pathways are presentin the reference proteome map. The wide coverage of theidentified proteins in the metabolic pathways indicates a goodapplication potential of using this proteome reference map tounderstand the metabolism-related physiology of C. acetobu-tylicum.

Solvent-tolerant strains were obtained through serial enrich-ment of liquid cultures with butanol. Strains SA-113 and G119

were developed in this manner and were tolerant to 15 and 18g/L butanol. Butanol tolerant mutant C. acetobutylicum SA-2derived from strain ATCC 824 by classic chemical mutagenesiscould grow in the presence of 18 g/L butanol.14 However, theSA-2 strain could only produce trace amount of butanol,14

suggesting the improved tolerance did not result in a propor-tional increase in butanol production. A C. beijerinckii strainBA101 generated by chemical mutagenesis has been shown toproduce 19 g/L butanol.61 Overexpression of spo0A also con-ferred increased tolerance and prolonged metabolism in re-sponse to butanol stress.55 In addition, overexpression ofgroESL in C. acetobutylicum has been shown to confer the hostincreased butanol tolerance and 17.1 g/L butanol was pro-duced.15 Moreover, the buk gene inactivated ATCC 824 straincould produce 16.7 g/L butanol.16 Furthermore, inactivationof solR gene, combined with increased adhE1 (previouslydesignated as aad) gene expression in ATCC 824 strain, resultedin a strain with improved butanol production (17.6 g/L).17 Thebutanol-tolerant mutant Rh8 that we developed after extensivemutagenesis and genome shuffling is able to tolerate 19 g/Lbutanol and can produce 15.3 g/L butanol. Although thebutanol titer produced by strain Rh8 is not the highest amongthe literatures, we were able to perform comparative proteomicanalysis between the mutant strain and the base strain of C.acetobutylicum, for which very limited proteomic data havebeen previously reported.

Comparative proteomic analysis between the wild type strainDSM 1731 and the mutant Rh8 in acidogenesis and solvento-genesis revealed a total of 102 differentially expressed proteins.The mechanism that strain Rh8 developed for an improvedbutanol tolerance can be seen from two aspects: upregulateproteins (e.g., chaperones and solvent formation related) inacidogenic phase, and further upregulate these proteins insolventogenic phase; or downregulate proteins (e.g., amino acidmetabolism and protein synthesis related) in acidogenic phase,and further downregulate these proteins in solventogenicphase. This suggests that strain Rh8 may have developed amechanism to prepare itself for coping with butanol challengesbefore butanol is produced, leading to an increased butanolproduction.

It has been previously reported that chaperone proteins playan important role in increasing solvent production and toler-ance of C. acetobutylicum.15 The well-characterized chaperoneproteins involved in butanol stress resistance include Hsp90,12,20

DnaK,12,20,55 GroES,12,20,55 GroEL,12,55 GrpE,20 Hsp18,12,20,55

YacI,20 ClpP,12,20 HtrA,20 and ClpC.12,20,55 Sixteen out of the 102differentially expression proteins identified in this study werechaperone proteins (Table 1). The 16 upregulated chaperoneproteins in Rh8 can be categorized into three groups: onlyupregulated in acidogenic phase (HtpG, GroEL, DnaK, andHtrA); or only in solventogenic phase (YacI, GroES, ClpP, Map,TufA, GreA, CAC1297, CAC0313, and CAC3598); or in bothacidogenic and solventogenic phase (Hsp18, GrpE, and ClpC).This resulted in a comparable or even higher total upregulationof chaperone proteins in strain Rh8 as compared with strainDSM 1731, which might contribute to the increased butanoltolerance of strain Rh8. These results suggest that increasedproduction of chaperone proteins might be a general conse-quence in C. acetobutylicum that can be either induced bybutanol stress20 or by adaptation to a high butanol concentra-tion as shown in this study.

Figure 6. Comparison of the expression of eleven genes at thelevel of mRNA and protein in the wild type strain DSM 1731 andthe mutant Rh8 in acidogenic phase. The ratio of expressedproteins between DSM 1731 and Rh8 has statistical significance(P < 0.05).

research articles Mao et al.

3058 Journal of Proteome Research • Vol. 9, No. 6, 2010

http://pubs.acs.org/action/showImage?doi=10.1021/pr9012078&iName=master.img-006.jpg&w=226&h=336

Interestingly, 5 out of the 16 chaperone proteins (TufA, GreA,CAC1297, CAC0313, CAC3598) were found for the first time inC. acetobutylicum that might contribute to the increasedbutanol tolerance. Within these 5 proteins, three elongationfactors, such as TufA, GreA, and N-terminal fragment ofelongation factor Ts (Tsf) seem to be involved in protein-proteininteraction with unfolded proteins.62 TufA interacts withunfolded and denatured proteins as do molecular chaperonesafter stress.62 In Streptococcus mutans, an increase expressionof TufA was found during acid-tolerant growth stage.63 GreAis one of the only few proteins that are upregulated during low-pH growth of Streptococcus mutans,63 and one of the only 9proteins that are up-regulated in Staphylococcus aureus inresponse to a challenge by cell wall-active antibiotic.64 In E.coli, overexpression of greA confers resistance to toxic levelsof divalent metal ions such as Zn2+ and Mn2+.65 Finally, Tsfhas been reported as a steric chaperone by functioning as astructural template for the correct folding of TufA.66 In E. coli,the protein denaturant guanidine hydrochloride stress-inducedincreased expression of Tsf.67 These findings suggest thatelongation factors may play important roles in butanol tolerance.

Ten out of the 102 differentially expression proteins in thisstudy are proteins involved in solvent formation (Table 1),which are known to increase expression at the onset ofsolventogenesis.21 The interesting observation is that 7 out of10 proteins (THL, AdhE1, CtfA/B, Adc, BdhA/B) in strain Rh8that are involved in acetone and butanol production signifi-cantly upregulated in acidogenic phase, resulting in a highertotal expression level in solventogenesis as compared to strainDSM 1731. We believe the significant upregulation of thesesolvent formation proteins contributed to the increased pro-duction of acetone and butanol in mutant Rh8. C. acetobutyli-cum strain ATCC 824 with GroESL when challenged with0.75%(v/v) butanol, the solvent formation genes thl, adhE1,ctfA/B, and bdhA/B were upregulated.20 The acids acetate,butyrate and butanol stress also upregulated solventogenicoperon adhE1-ctfA-ctfB.12 This suggests that solvent formationproteins are not only regulated by short-term butanol stress,but can also be regulated by long-term adaptation to a higherbutanol concentration.

Twenty-five proteins out of 102 differentially expressionproteins are involved in amino acid metabolism and proteinsynthesis (Table 1), which represents the largest functionalgroup. The majority of these proteins were grouped in Cluster3 of Figure 5, showing that Rh8 might develop a mechanismto slow down amino acid metabolism and protein synthesis toadapt to butanol challen