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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: [email protected]. 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
Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant research articles
Journal of Proteome Research • Vol. 9, No. 6, 2010 3047
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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3048 Journal of Proteome Research • Vol. 9, No. 6, 2010
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
Sery
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
-Ph
osp
ho
glyc
erat
ed
ehyd
roge
nas
egi
|150
2283
433
283/
6.17
CA
C00
15se
rA86
40/7
1-
160
NS
-2.
0(1
.67
×10
-2 )
-4.
1(3
.39
×10
-5 )
36R
elat
edto
HT
Hd
om
ain
of
Spo
OJ/
Par
A/P
arB
/rep
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
-am
ino
-aci
dtr
ansa
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
×10
-3 )
Mem
bra
ne
tran
spo
rt52
Iro
n-r
egu
late
dA
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
/58
-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
S2.
4(7
.77
×10
-3 )
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
-3 )
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
-4 )
1.4
(9.0
4×
10-
3 )2.
7(6
.00
×10
-4 )
72M
ole
cula
rch
aper
on
eD
naK
,H
SP70
fam
ily
gi|1
5024
210
6560
9/4.
80C
AC
1282
dn
aK48
549
21.
4(3
.50
×10
-3 )
NS
NS
73A
TP
ase
wit
hch
aper
on
eac
tivi
tycl
pC
,tw
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×
10-
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
S1.
9(1
.04
×10
-3 )
1.5
(1.6
3×
10-
3 )
75H
trA
-lik
ese
rin
ep
rote
ase
(wit
hP
DZ
do
mai
n)
gi|1
5025
449
4555
6/6.
02C
AC
2433
htr
A54
116
42.
3(9
.10
×10
-4 )
NS
NS
76C
o-c
hap
ero
nin
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
.71
×10
-3 )
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
.64
×10
-3 )
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
.49
×10
-2 )
85C
ell
div
isio
nG
TP
ase
Fts
Zgi
|150
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
(1.4
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
-3 )
88C
hem
ota
xis
pro
tein
Ch
eBgi
|150
2522
038
121/
8.86
CA
C22
22ch
eB46
317
6N
SN
S2.
7(1
.80
×10
-3 )
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
.78
×10
-2 )
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
(9.1
1×
10-
3 )-
2.6
(5.2
4×
10-
3 )N
S
91F
usi
on
of
Uro
po
rph
yrin
oge
n-I
IIm
eth
ylas
ere
late
dp
rote
inan
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
.90
×10
-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
.47
×10
-4 )
95P
uta
tive
met
hyl
tran
sfer
ase
gi|1
5023
435
2420
6/5.
50C
AC
0567
364
243
NS
NS
3.9
(1.0
7×
10-
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
|150
2528
743
200/
7.2
CA
C22
82tg
t38
387
NS
3.9
(2.0
8×
10-
3 )8.
7(1
.25
×10
-2 )
99P
red
icte
den
zym
ew
ith
TIM
-bar
rel
fold
gi|1
5022
975
2459
6/8.
99C
AC
0148
434
257
NS
7.1
(5.7
5×
10-
4 )10
.9(2
.36
×10
-3 )
100
hyp
oth
etic
alp
rote
inC
AC
0488
gi|1
5023
348
1350
6/5.
05C
AC
0488
411
111
2.7
(1.1
5×
10-
3 )N
SN
S10
1H
ypo
thet
ical
pro
tein
,C
F-2
1fa
mil
ygi
|150
2318
019
463/
5.09
CA
C03
3439
317
0N
S2.
7(2
.01
×10
-3 )
NS
102
Hyp
oth
etic
alp
rote
inC
AC
1171
gi|1
5024
092
2097
8/5.
54C
AC
1171
543
165
NS
1.7
(2.5
9×
10-
2 )3.
2(1
.39
×10
-2 )
aP
rote
inid
enti
fica
tio
nn
um
ber
s(S
po
tsID
)co
rres
po
nd
toth
en
um
ber
sin
Fig
ure
S3o
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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
http://pubs.acs.org/action/showImage?doi=10.1021/pr9012078&iName=master.img-005.jpg&w=498&h=420
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