Proteome Reference Map and Comparative Proteomic Analysis

Apr 29, 2010 - Proteome Reference Map and Comparative Proteomic Analysis between a Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant with E...
1 downloads 10 Views 3MB Size
Proteome Reference Map and Comparative Proteomic Analysis between 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 of Sciences, 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 of California, Los Angeles, California 90095 Received December 29, 2009

The solventogenic bacterium Clostridium acetobutylicum is an important species of the Clostridium community. 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 spots representing 564 different proteins by mass spectrometry, covering approximately 50% of major metabolic pathways. To better understand the relationship between butanol tolerance and butanol yield, we performed a comparative proteomic analysis between the wild type strain DSM 1731 and the mutant Rh8, which has higher butanol tolerance and higher butanol yield. Comparative proteomic analysis of two strains at acidogenic and solventogenic phases revealed 102 differentially expressed proteins that are mainly involved in protein folding, solvent formation, amino acid metabolism, protein synthesis, nucleotide metabolism, transport, and others. Hierarchical clustering analysis revealed that over 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 protein synthesis related) in both acidogenic and solventogenic phase, which, respectively, are only upregulated or downregulated in solventogenic phase in the wild type strain. This suggests that Rh8 cells have evolved a mechanism to prepare themselves for butanol challenge before butanol is produced, leading to an increased butanol yield. This is the first report on the comparative proteome analysis of a mutant strain and a base strain of C. acetobutylicum. The fundamental proteomic data and analyses will be useful for further elucidating the biological mechanism of butanol tolerance and/or enhanced butanol production. 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, Grampositive, spore-forming, obligate anaerobe that is capable of fermenting a wide variety of sugars (e.g., glucose, galactose, cellobiose, mannose, xylose, and arabinose) to acids (acetic acid and butyric acid) and solvents (acetone, butanol, and ethanol).1 C. acetobutylicum culture was extensively used to produce acetone and butanol from starch for industrial purposes.2 * To whom correspondence should be addressed. Yin Li, Institute of Microbiology, Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District, Beijing 100101, China. E-mail: [email protected]. Tel: +8610-64807485. 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 Published on Web 04/29/2010

Recently, anaerobic fermentative production of butanol using this bacterium gained remarkable interest as butanol is considered as a potential superior biofuel alternative.3 Recent developments in genetics,4 genomics,5 and proteomics6,7 of C. acetobutylicum have greatly increased our understanding of the solvent production physiology, which is extremely important for improving butanol production by means of metabolic engineering or systems biotechnology. Butanol toxicity is the major barrier for cost-effective fermentative production of butanol. This can be seen from the fact that C. acetobutylicum fermentations rarely produce butanol higher than 13 g/L, a level that is inhibitory for the growth of C. acetobutylicum and is generally considered as the toxic limit.8 Butanol toxicity in C. acetobutylicum is quite severe, and this has been attributed to the chaotropic effect on cell membrane9,10 and the inhibition effects on nutrient transport, glucose uptake, and membrane-bound ATPase activity.9 Mod10.1021/pr9012078

 2010 American Chemical Society

Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant erate increases in butanol titers have been shown to elicit a response similar to a heat shock.11 Butanol-stressed exponentially growing C. acetobutylicum resulted in upregulation of numerous chaperone genes (dnaK, groES, groEL, hsp90, hsp18, clpC, and htrA), solventogenic genes, and glycerol metabolism genes glpA and glpF.12 Butanol-tolerant strains have been developed from C. acetobutylicum by using classic chemical mutagenesis, continuous culture, serial enrichment procedures, and targeted genetic/metabolic engineering.13–18 The improved butanol tolerance has different impacts on butanol production (decrease,14,18 minor improvement,13,19 or significant (>20%) improvement),15–17 suggesting the relationship between butanol tolerance and butanol production is rather complex and remains largely unknown. The availability of the complete genome sequence of C. acetobutylicum5 enables the genome-wide investigation of the mechanism for butanol tolerance. Genome-wide transcriptome studies on C. acetobutylicum were performed.12,20,21 Characterization of the C. acetobutylicum Spo0A mutant SKO1 by DNA microarray analysis revealed that Spo0A inactivation triggered down-regulation of solventogenic, sporulation, and carbohydrate metabolism genes and up-regulation of flagellar and chemotaxis genes.22 Transcriptome analysis of C. acetobutylicum mutant 824 (pGROE1) with overexpressed groESL revealed an increased expression of motility and chemotaxis genes.15 Proteomics is a powerful tool to understand the cellular status at the protein level, which cannot be deciphered from either genome or transcriptome analysis. Proteomics approaches are increasingly employed to identify proteins that can be used as targets for metabolic engineering.23–25 Since two-dimensional electrophoresis (2-DE) was introduced as a tool to separate complex mixtures of cellular proteins,26 a large number of prokaryotic proteomes have been studied.27–29 Studies on C. acetobutylicum proteomics are underway. A proteomic analysis of the C. acetobutylicum during the transition from acidogenesis to solventogenesis discovered some changes in the protein pattern.6 A comparative proteomic analysis between C. acetobutylicum wild type strain ATCC 824 and its mutant 824(pMSPOA) discovered that Spo0A overexpression affected the abundance of proteins involved in glycolysis, translation, heat shock stress response, and energy production.7 The aim of this study was to establish a comprehensive cytoplasmic proteome reference map for C. acetobutylicum so that the reference map can be used as a fundamental tool for comparative proteomics study to further increase our knowledge on the physiology of this species. To illustrate the significance of this reference map, we carried out a comparative proteomic analysis between two strains, C. acetobutylicum wild type strain DSM 1731 (butanol tolerance 13 g/L) and its mutant Rh8 (butanol tolerance 19 g/L), with the aim to obtain fundamental data for understanding the biological mechanism on butanol tolerance and/or butanol yield.

Experimental Section Bacteria and Culture Conditions. C. acetobutylicum DSM 1731, obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), was used for generating the proteome reference map. Mutant Rh8 is a genome-shuffled strain derived from strain DSM 1731 and its butanol-tolerant mutants. Cells from a single colony were used to inoculate liquid reinforced clostridial medium (RCM),30 which were then heat shocked at 75 °C for 10 min before growing at 37 °C. When the OD600 reached 1.0, the culture was

research articles

stored at 30 °C for one week to produce spores.31 Spore counts were measured as following: 100 µL seed cultures were mixed with 900 µL deionized distilled water. Disposable micropipets were used to stir the mixture and drops of the suspensions were transferred to a hemacytometer slide. An estimate of the total number of spores produced in each chamber was derived arithmetically from the hemacytometer counts.32 Precultures (10 mL) of strains DSM 1731 and Rh8 were inoculated to about 106-107 spores and pasteurized for 10 min at 75 °C before incubation, then cultured in a 250 mL bottle containing 100 mL of clostridial growth medium (CGM)33 at 37 °C for 12 h. In all experiments, cell growth was monitored by measuring the absorbance of the culture broth at 600 nm (OD600) with a model DU series 800 spectrophotometer (Beckman, Fullerton, CA). For proteomic analysis, cells were harvested at exponential growth phase at OD600 of 2.0, which is corresponding to 6.5 × 108 colony forming units/mL. Generation of Mutant Rh8. Genome shuffling34 was used to generate C. acetobutylicum mutant Rh8, which is more tolerant to butanol. C. acetobutylicum DSM 1731 was cultured in reinforced clostridial medium (RCM)30 at 37 °C for 12 h. Cells were harvested at acid-production phase (OD600 ) 2.0) and resuspended in 0.1 M potassium phosphate buffer (pH 7.0) containing 1% (v/v) diethyl sulfate (DES). The mixture was incubated at 37 °C for 15 min, and the cells were washed twice with Trypticase-Glucose-Yeast extract medium (TGY)35 and then resuspended in RCM and grown at 37 °C for 48 h. The cultures were plated on RCM agar (RCM + 2% agar) containing various amounts of butanol and incubated at 37 °C for 48 h. Colonies grown on RCM agar containing 18 g/L butanol were selected. Four stable mutants which can tolerate 18 g/L butanol were subjected to protoplast fusion. Each mutant was grown in clostridial basal medium (CBM)36 containing 0.5% (w/v) glycine at 37 °C for 24 h. Cells were harvested by centrifugation at 3000× g for 10 min, washed twice with isotonic buffer (CBM containing 0.5 M sucrose), and treated with isotonic buffer containing 1 mg/mL lysozyme at 37 °C for 20 min. Protoplasts were harvested by centrifugation at 1000× g for 15 min and gently washed with isotonic buffer. Protoplasts from different mutants were mixed and divided equally into two parts. One part was inactivated with UV for 20 min, and the other part was inactivated by heating at 80 °C for 30 min. Both inactivated protoplasts preparations were mixed and fused by suspension in 5 mL isotonic buffer containing 40% PEG 6000 at room temperature for 5 min. Dilutions of the fused protoplasts were plated onto a regeneration medium37 and incubated at 37 °C for 48 h. Colonies grown on the regeneration medium were selected to test their tolerance to butanol and the butanol production capability. A progeny cell culture that can grow in the medium containing 19 g/L butanol was obtained. Fermentation Experiments. Batch fermentation experiments of C. acetobutylicum DSM 1731 and its mutant Rh8 were carried out in BioFlo 110 fermentors (New Brunswick Scientific, Edison, NJ) containing 4.0 L (working volumes) of CGM,33 according to the cultivation method described in the literature38 with slight modification. Briefly, 200 mL seed cultures were inoculated into a fermentor containing 4 L CGM. The initial pH of the fermentation was 6.5, and the pH was allowed to drop to 5.0 as the culture progressed. Subsequently, the pH was automatically maintained at or above 5.0 by adding 6 M ammonium hydroxide. The concentrations of the main metabolites in the cell-free fermentation broth (acetate, butyrate, acetone, butanol, ethanol, and glucose) were determined using Journal of Proteome Research • Vol. 9, No. 6, 2010 3047

research articles an Agilent 1200 high performance liquid chromatography (Agilent Technologies, Santa Clara, CA). A Bio-Rad Aminex HPX-87H ion exchange column (7.8 by 300 mm) (Bio-Rad Laboratories, Inc., Richmond, CA) was used with a mobile phase of 0.05 mM sulfuric acid at 15 °C with a flow rate of 0.50 mL/min. A refractive index (RI) detector (Agilent) was used at 30 °C for signal detection. Butanol Challenge Experiments. Strain DSM 1731 and its mutant Rh8 were grown in 500 mL flasks containing 400 mL CGM at 37 °C statically. When OD600 of 1.0 ( 0.05 was achieved (approximately 12 h), each culture was split into four 100 mL aliquots and challenged with 0, 6, 12, and 18 g/L butanol. The growth of these two strains in the presence of different concentrations 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 harvested by centrifugation at 10 000× g at 4 °C for 10 min and washed three times with 45 mL of ice-cold TE buffer (10 mM Tris-HCl, 5 mM EDTA, pH 7.5). The resulting C. acetobutylicum DSM 1731 and Rh8 cell pellets (about 0.80 g wet weight) were resuspended in 10 mL of lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS (w/v), 50 mM DTT, and 10 mM PMSF) containing a complete protease inhibitors cocktail (Roche diagnostics, Mannheim, Germany). The cells were sonicated on ice for 15 min using a Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT) with the following conditions: 5 s of sonication with a 5-s interval, set at 50% duty cycle. After adding 10 µg/ mL nuclease mix (GE Healthcare, Uppsala, Sweden), the cell lysate was incubated at ambient temperature for 30-45 min to degrade nucleic acids. The resulting lysate was collected and centrifuged at 150 000× g in a 90 Ti rotor (Beckman, Fullerton, CA) at 4 °C for 45 min. The supernatant was diluted with three volume of ice cold acetone. After mixing and incubation at -20 °C for 16 h, proteins were sedimented by centrifugation at 15 000× g at 4 °C for 30 min. The protein precipitants were solubilized in sample lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS, 0.5% Pharmalyte pH 3-10). Protein concentration was measured 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. The extracted proteins from both C. acetobutylicum strain DSM 1731 and Rh8 were subjected to 2-DE, respectively. IEF was performed on Ettan IPGphor 3 system (GE Healthcare, Uppsala, Sweden) using IPG strips (GE Healthcare). For IPG strips of pH 4-7, approximately 1 mg of protein per sample in rehydration buffer (8 M urea, 2 M thiourea, 4% CHAPS, 0.5% Pharmalyte pH 4-7, and 0.001% bromophenol blue) was run using the ingel sample rehydration technique according to the manufacturer’s instructions for IEF. IEF was performed using the following voltage program: 30 V constant for 12 h, gradient to 200 V for 4 h, gradient to 1000 V within 4 h, gradient to 10 000 V within 4 h then 10 000 V for 4 h for a total of 65 000 V · h. For IPG strips of pH 6-11, approximately 1 mg protein per sample (100 µL) was loaded on a previously rehydrated strip (rehydrated for 12 h) by anodic cup loading as recommended by the manufacturer. IEF was performed using the following voltage program slightly modified from previously reported:39 150 V constant for 4 h, gradient to 300 V within 2 h, gradient to 600 V within 2 h, gradient to 8000 V within 30 min and then 8000 V until a total of 32 000 V · h had been achieved. The temperature was maintained at 20 °C for IEF. 3048

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

Mao et al. After completion of the first-dimension IEF, each strip was equilibrated in 10 mL equilibration buffer 1 (6 M urea, 50 mM DTT, 30% glycerol, 50 mM Tris-HCl, pH 8.8) for 15 min and then 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 subsequently placed on the top of 12.5% SDS-PAGE gels. A denaturing solution (0.7% agarose, 0.1% SDS, 192 mM glycine, 25 mM TrisHCl (pH 8.8), 0.001% bromophenol blue) was loaded onto the gel strips. After agarose solidification, electrophoresis was performed 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 by 5-6 h at 10 W/gel until the bromophenol blue reached the bottom. Six gels were run in parallel on Ettan DALTsix electrophoresis system (GE Healthcare). For each condition, 2-DE experiments were carried out in triplicate. The protein spots were 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 dpi resolution using ImageScannerIII (GE Healthcare). Comparative analysis of the protein spots was performed using Image Master 6.0 2-D platinum software (GE Healthcare) as previously described.41 All images were submitted to automatic spot detection according to the manufacturer’s recommendations. The spots were checked manually to eliminate any possible artifacts including background noise and streaks. To obviate batch-to-batch variance, spots that were consistently reproducible in all gel images, including both the biological and technical replicates, were chosen for subsequent analyses. All images were aligned and matched by using the common spots present in all images as landmarks, to detect potential differentially expressed proteins. Spot normalization was performed using relative volumes (%Vol) to quantify and compare the gel spots as described previously,41 with the aim to make the data independent of the experimental variations between gels. Only protein spots showing reproducible changes in protein abundance, by multiple experiments (at least three biological repetitions and two technical replicates), were considered as biomarkers associated with wild type strain and mutant. Statistical analysis was performed using Students t-test. P < 0.05 was considered significant. Protein Expression Profile Analysis. Hierarchical clustering analysis was used to group proteins exhibiting similar expression profiles. The relative volumes (%Vol) of each protein spot, obtained from the Image Master 6.0 2-D platinum software (GE Healthcare), were used for hierarchical cluster analysis. The protein profile normalization was managed as described previously.42 The differentially expressed proteins were grouped on the basis of similarity in expression profile by using KMC support (TIGR MeV, version 4.5.1).43 In-Gel-Digestion. All protein spots were excised and submitted to in-gel-digestion as previously described41 with slight modifications. Briefly, the Coomassie blue-stained protein spots were manually excised using a spot picker (The Gel Company, San Francisco, CA). The spots were transferred to Eppendorf tubes, sealed, and stored at -80 °C until further processing. One-hundred microliters of 50% ACN solution containing 50 mM ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO) were added to each tube, and the mixtures were incubated with occasional vortexing for 30 min. This process was repeated until all gel spots were completely destained. The spots were then dehydrated with 100 µL of ACN at room temperature for 15 min. After ACN removal, the gel spots were dried under vacuum

Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant and rehydrated in 20 µL of proteomics sequencing grade trypsin (10 ng/µL; Cat.: T6567, Sigma-Aldrich, St. Louis, MO), in which lysine residues have been reductively methylated, leading to resistance to autolysis. After rehydration at 4 °C for 45 min, excess trypsin solution was removed, and 10 µL of 50 mM ammonium bicarbonate was added followed by incubation at 37 °C for 16 h. Peptides were extracted twice by the addition of 30 µL of 5% formic acid (v/v)/50% ACN (v/v) solution and vortexing for 30 min. The peptide samples were concentrated by using Speed Vac (Thermo Savant, Waltham, MA) to approximately 10 µL and stored at -20 °C for mass spectrometric analysis. MALDI-TOF MS and MS/MS Analysis. A 0.4 µL aliquot of the concentrated tryptic peptide mixture in 0.1% TFA was mixed with 0.4 µL of CHCA matrix solution (5 mg/mL CHCA in 50% ACN/0.1% TFA) and spotted onto a freshly cleaned target plate. After air drying, the crystallized spots were analyzed on the Applied Biosystems 4700 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems, Framingham, MA). MS calibration was automatically performed by a peptide standard Kit (Applied Biosystems) containing des-Arg1-bradykinin (m/z 904), 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 peaks of Glu1-fibrinopeptide B. All MS mass spectra were recorded in the reflector positive mode using a laser operated at a 200 Hz repetition rate with wavelength of 355 nm. The accelerated voltage was operated at 2 kV. The MS/MS mass spectra were acquired by the data dependent acquisition method with the 10 strongest precursors selected from one MS scan. All MS and MS/MS spectra were obtained by accumulation of at least 1000 and 3000 laser shots, respectively. Neither baseline subtraction nor smoothing was applied to recorded spectra. MS and MS/ MS data were analyzed and peak lists were generated using GPS Explorer 3.5 (Applied Biosystems). MS peaks were selected between 850 to 3700 Da and filtered with a signal-to-noise ratio greater than 20. A peak intensity filter was used with no more than 50 peaks per 200 Da. MS/MS peaks were selected based on a signal-to-noise ratio greater than 10 over a mass range of 60 to 20 Da below the precursor mass. MS and MS/MS data were analyzed using MASCOT 2.0 search engine (Matrix Science, London, U.K.) to search against the C. acetobutylicum protein sequence database (10 159 sequences; 3 116 366 residues) downloaded from NCBI database on March 20 2008. Searching parameters were as follows: trypsin digestion with one missed cleavage, variable modifications (oxidation of methionine and carbamidomethylation of cysteine), and the mass tolerance of precursor ion and fragment ion at 0.2 Da for +1 charged ions. For all proteins successfully identified by Peptide Mass Fingerprint and/or MS/MS, Mascot score greater than 53 (the default MASCOT threshold for such searches) was accepted as significant (p value 5.0) of (A) the wild type strain DSM 1731 and (B) the mutant Rh8. Arrows indicate samples were withdrawn for proteome analysis. (C) Glucose utilization profiles; (D) pH profiles; (E) average butanol yield profiles; (F) average butanol productivity profiles. DSM 1731 (open symbols) and Rh8 (solid symbols).

folding (16 proteins), solvent formation (10), amino acid metabolism (12), protein synthesis (11), nucleotide metabolism (9) transport (7), and others. Of the 102 proteins, the genes coding for 52 proteins were reported to respond to butanol stress or the transition from acidogenesis to solventogenesis,6,12,15,20,21,55,56 while the other 50 proteins have not been previously reported to be related to butanol tolerance or the transition from acidogenesis to solventogenesis (labeled as

asterisk in Figure 5). We compared the profiles of differentially expressed proteins of C. acetobutylicum DSM 1731 and Rh8 with the profiles of differentially expressed genes of C. acetobutylicum ATCC 82456 (Figure S5 of Supporting Information 1). The results showed that only part of the DNA microarray data overlapped with the differentially expressed protein that we identified. To explore the difference between the proteomes of strains DSM 1731 and Rh8 during acidogenesis and solventogenesis, Journal of Proteome Research • Vol. 9, No. 6, 2010 3053

3054

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

Threonyl-tRNA synthetase Prolyl-tRNA synthetase Glycyl-tRNA synthetase Glutamyl-tRNA synthetase Aspartyl-tRNA synthetase Methionyl-tRNA synthetase Seryl-tRNA synthetase Aminotransferase D-3-Phosphoglycerate dehydrogenase Related to HTH domain of SpoOJ/ ParA/ParB/repB family Dihydroxy-acid dehydratase Isopropylmalate dehydrogenase 3-isopropylmalate dehydratase, small subunit UDP-N-acetylmuramyl tripeptide synthase, MURE Glutamine synthetase type III

27 28 29 30 31 32 33 34 35

41

40

37 38 39

36

Exopolyphosphatase Ferredoxin-nitrite reductase Flavoprotein flavodoxin Riboflavin synthase beta-chain NifU homologue involved in Fe-S cluster formation

glucose-6-phosphate isomerase Phosphomannomutase Transketolase Pyruvate kinase (pykA) Acetolactate synthase large subunit Pyruvate-formate lyase Acetyl-CoA acetyltransferase (thiolase A) Acetate kinase bifunctional acetaldehyde-CoA/ alcohol dehydrogenase Butyrate-acetoacetate COA-transferase subunit A (CtfA) Butyrate-acetoacetate COA-transferase subunit B (CtfB) Acetoacetate decarboxylase Phosphate butyryltransferase NADH-dependent butanol dehydrogenase B (BDH II) NADH-dependent butanol dehydrogenase A (BDH I) Aconitase A Biotin carboxyl carrier protein of acetyl-CoA carboxylase Malic enzyme Pyruvate:ferredoxin oxidoreductase Tetrahydrofolate dehydrogenase/ cyclohydrolase

protein description

b

21 22 23 24 25 26

18 19 20

16 17

15

12 13 14

11

10

8 9

1 2 3 4 5 6 7

match id

a

gi|15025687

gi|15025861

gi|15026238 gi|15026239 gi|15026240

gi|15022835

gi|15025373 gi|15026247 gi|15026266 gi|15023897 gi|15025273 gi|15026047 gi|15022836 gi|15022833 gi|15022834

gi|15025130 gi|15022918 gi|15023938 gi|15025470 gi|15023462 gi|15026370

gi|15024543 gi|15025228 gi|15025071

gi|15023877 gi|15026668

gi|15026377

gi|15004868 gi|15026139 gi|15026376

gi|15004867

gi|15004866

gi|15024711 gi|15004865

gi|15025711 gi|15025346 gi|15023846 gi|15023381 gi|15026237 gi|15023887 gi|15025920

NCBI accession number

76694/5.56

53811/5.52

58329/6.02 38955/5.01 18013/5.74

47693/5.78

73150/5.68 64096/5.37 53343/5.30 55436/5.41 68200/5.24 73571/5.57 48622/5.98 39952/6.23 33283/6.17

34674/5.42 58881/6.44 44462/5.23 15611/4.80 16476/6.14 15918/4.91

59450/5.78 128600/5.88 30669/6.34

69590/5.83 17845/4.50

43183/5.81

27690/5.81 32207/6.34 43259/5.75

23666/7.79

23797/8.99

44313/6.23 95774/8.44

49759/5.37 64674/5.59 72662/5.21 50560/5.74 60075/5.35 84406/6.24 41443/6.92

theor. Mrc/ pId

CAC2658 glnA

CAC2819 murE

CAC3170 ilvD CAC3171 leuB CAC3172 leuD

CAC0016

36

67

40 45 32

43

21/34

399 113

-

82 194 122

172

211 107 471 416 62 203 541 626 160

416 689 171 291 338 203

372 687 98

4

3 6 2

3

1 3 7 5 7 6 -

7 6 4

97 310

6

Amino acid and protein synthesis-related proteins CAC2362 thrS 43 CAC3178 proS 38 CAC3195 glyS 63 CAC0990 gltX 59 CAC2269 aspS 30 15/39 CAC2991 metS 62 41/58 CAC0017 serS 60 CAC0014 serC 71 CAC0015 serA 86 40/71

16/33

72

314 612 432

132

580

509 113

114 169 97 368 210 490 220

1

5 5 5

1

4

5 -

4 6 5 3 3

64 41 46

33 74

31

67 71 58

36

82

23/68

6 1 6 1

1 -

Energy metabolism CAC2138 75 CAC0094 74 CAC1027 61 45/69 CAC2452 64 CAC0593 ribH 66 CAC3292 69

CAC1589 malS CAC2229 CAC2083 folD

CAC0971 citB CAC3572 accB

CAC3299 bdhA

CAP0165 adc CAC3076 ptb CAC3298 bdhB

CAP0164 ctfB

CAP0163 ctfA

60 26

Carbohydrate metabolism pgi 61 45 34/69 tkt 37 20/33 pykA 60 ilvB 46 pflB 55 thl 62

CAC1743 askA CAP0162 adhe1

CAC2680 CAC2337 CAC0944 CAC0518 CAC3169 CAC0980 CAC2873

gene locus

ratiog (P-value)

NS

NS

NS NS NS

NS

-2.3 (3.17 × 10-4) NS NS NS NS NS NS NS NS

2.3 (1.37 × 10-3) -1.5 (1.87 × 10-2) 1.7 (4.70 × 10-5) NS -1.7 (1.81 × 10-2) NS

NS NS NS

-1.9 (1.36 × 10-2)

NS NS -2.9 (7.69 × 10-4)

7.7 (4.07 × 10-5)

-4.9 (8.20 × 10-5) -2.6 (4.79 × 10-4) -2.6 (2.28 × 10-4) -2.4 (1.52 × 10-4) -5.8 (8.22 × 10-3) -2.6 (2.11 × 10-3) -2.6 (2.52 × 10-5) -2.6 (8.79 × 10-3) -4.1 (3.39 × 10-5)

-2.2 (4.90 × 10-2) -2.7 (6.99 × 10-3) NS 9.0 (3.93 × 10-6) 2.9 (1.08 × 10-3) NS

-2.4 (1.01 × 10-2) 2.0 (1.58 × 10-2) 2.5 (2.96 × 10-3)

-4.8 (2.65 × 10-4) -6.9 (3.29 × 10-2)

-8.4 (3.74 × 10-3) NS

NS NS NS

-2.6 (5.57 × 10-4) -1.9 (3.20 × 10-2)

-6.0 (5.99 × 10-4) -2.5 (3.23 × 10-4) -2.6 (4.12 × 10-4) NS -6.1 (3.20 × 10-3) NS -2.0 (2.54 × 10-2) -1.5 (4.41 × 10-2) -2.0 (1.67 × 10-2)

NS -2.7 (8.50 × 10-4) NS NS NS 2.8 (2.33 × 10-3)

-2.5 (4.87 × 10-2) -6.0 (1.00 × 10-5) -2.2 (4.33 × 10-3) NS 2.5 (3.17 × 10-4) 1.5 (3.29 × 10-2)

-2.1 (1.27 × 10-2) -2.4 (7.75 × 10-4) 1.3 (4.90 × 10-3) 2.1 (8.39 × 10-3)

NS

3.2 (4.48 × 10-3) NS NS

2.5 (2.44 × 10 ) 2.4 (2.77 × 10-4) NS 2.7 (1.28 × 10 ) NS 4.1 (3.76 × 10-4)

-3

15.6 (3.06 × 10-5) -3

2.7 (9.57 × 10-3)

2.7 (9.90 × 10-3)

3.6 (1.84 × 10-3)

2.7 (3.54 × 10-3)

NS 2.5 (2.40 × 10-4) -3.8 (2.48 × 10-4) -3.2 (2.57 × 10-5) NS -12.3 (9.96 × 10-3) NS

-1.7 (2.74 × 10-2) -2.0 (1.59 × 10-3) 5.7 (3.42 × 10-4) 3.5 (1.87 × 10-2)

1.8 (2.70 × 10-3) NS -3.3 (5.12 × 10-4) -2.3 (1.08 × 10-2) NDh -3.4 (5.38 × 10-4) NS

NS 1.7 (1.21 × 10-2)

2.1 (2.31 × 10-3) 1.4 (1.85 × 10-3) NS NS NDh NS 2.0 (2.43 × 10-4)

unique DSM 1731/ peptides peptides Rh8 solventogenesis/ Rh8 squence matched/ detected MASCOT acidogenesis/DSM DSM 1731 solventogenesis/ e f gene coverage (%) searched by MS/MS score 1731 acidogenesis acidogenesis Rh8 acidogenesis

Table 1. Functional Classification of Different Proteins in C. acetobutylicum DSM 1731 and its Mutant Rh8

research articles Mao et al.

76 77

75

74

73

70 71 72

68 69

61 62 63 64 65 66 67

60

59

58

57

56

55

54

53

52

48 49 50 51

47

46

43 44 45

42

match id

a

b

Molecular chaperone, HSP90 family Molecular chaperone (small heat shock protein), HSP18 Chaperonin GroEL, HSP60 family Molecular chaperone GrpE Molecular chaperone DnaK, HSP70 family ATPase with chaperone activity clpC, two ATP-binding domain Arginine kinase related enzyme, YACI B.subtilis ortholog HtrA-like serine protease (with PDZ domain) Co-chaperonin GroES, HSP10 family Rubrerythrin

GTPase, sulfate adenylate transferase subunit 1 Polyribonucleotide nucleotidyltransferase CTP synthase Orotate phosphoribosyltransferase Adenine phosphoribosyltransferase Nudix (MutT) family hydrolase Uridylate kinase DNA polymerase III beta subunit RECA recombinase, ATPase

Iron-regulated ABC transporter ATPase subunit (SufC) ABC-type polar amino acid transport system, ATPase component FoF1-type ATP synthase alpha subunit FoF1-type ATP synthase beta subunit FoF1-type ATP synthase gamma subunit ABC transporter, ATP-binding protein Na+-ABC transporter (ATP-binding protein), NATA

Glu-tRNAGln amidotransferase subunit A Glycine hydroxymethyltransferase DAHP synthase related protein Cysteine synthase/cystathionine beta-synthase, CysK Branched-chain-amino-acid transaminase UDP-N-acetylglucosamine pyrophosphorylase Ribosomal protein L4 SpoOJ regulator, soj/para family Ferric uptake regulation protein Ribosome-associated protein Y (PSrp-1)

protein description

Table 1. Continued

45556/6.02 10420/5.06 20094/5.50

gi|15025449 gi|15025737 gi|15026696

38785/5.42

91978/5.94

58038/4.89 22705/4.50 65609/4.80

72361/5.09 17734/5.27

60042/5.42 25439/5.68 18957/4.94 19855/5.76 25837/5.82 41088/4.83 38179/6.88

77940/5.28

58919/5.74

26659/6.56

35696/6.97

31175/9.19

51062/4.87

55198/5.21

27293/6.93

27422/5.83

22851/9.78 28618/5.42 17711/5.59 20281/5.59

49661/6.01

37488/5.94

45583/6.16 37191/6.16 32918/5.54

53175/5.47

theor. Mrc/ pId

gi|15026260

gi|15026259

gi|15025736 gi|15024209 gi|15024210

gi|15026394 gi|15026820

gi|15025939 gi|15022847 gi|15025279 gi|15024831 gi|15024761 gi|15022819 gi|15024789

gi|15024781

gi|15022935

gi|15026646

gi|15023891

gi|15025912

gi|15025911

gi|15025913

gi|15023775

gi|15026366

gi|15026198 gi|15004880 gi|15024645 gi|15025892

gi|15026295

gi|15024425

gi|15025267 gi|15023790 gi|15025235

gi|15025700

NCBI accession number

49

52

47

77

smbA dnaN recA

ctrA pyrE apt

42 61 88 50 61 62 45

30

CAC2704 groES CAC3598

CAC2433 htrA

CAC3190 yacI

CAC3189 clpC

CAC2703 groEL CAC1281 grpE CAC1282 dnaK

86 76

54

58

22

49 58 48

Chaperone proteins CAC3315 htpG 47 CAC3714 hsp18 69

CAC2892 CAC0027 CAC2275 CAC1854 CAC1789 CAC0002 CAC1815

CAC1808 pnpA

30/67

22/30

Nucleotide metabolism CAC0110 cysN 43 26/58

CAC3551 natA

CAC0984

CAC2866 atpG

CAC2865 atpD

47

45

5 5

1

5

3

1 5

383 400

164

544

340

70 154 492

277 466

151 142 380 79 393 306 113

3 5 2 5 6 1 2 4

239

90

205

109

197

795

632

79

503

442 264 197 339

226

63

373 124 158

355

2

-

2

1

2

7

6

3

5

5

5 -

Membrane transport 81

CAC2867 atpA

CAC0879

CAC3288

24/68

35/70 34/59

6

4 4 5 6

47

59

59 73 70

44

68 43 83 59

CAC3132 rplD CAP0177 CAC1682 CAC2847

CAC3222 gcaD

CAC1479 ilvE

CAC2264 glyA CAC0892 CAC2235 cysK

CAC2670

gene locus

ratiog (P-value)

-3

1.5 (1.63 × 10-3)

NS NS

2.3 (9.10 × 10 )

-4

1.9 (1.04 × 10-3) NS

6.9 (1.22 × 10-4) NS

1.5 (1.77 × 10-2)

NS 2.3 (3.99 × 10-3) 1.5 (3.02 × 10-2)

NS 2.2 (3.65 × 10-3) NS

NS 2.7 (6.00 × 10-4) NS

1.7 (7.35 × 10-3) 1.4 (9.04 × 10-3) NS

1.7 (1.23 × 10-3) 1.5 (6.00 × 10-4) 1.4 (3.50 × 10-3)

NS 2.4 (1.25 × 10-6)

-4.1 (1.11 × 10-3) 1.4 (6.17 × 10-3) 2.0 (9.53 × 10-3) 2.4 (6.38 × 10-3) 3.4 (4.58 × 10-3) 2.1 (1.24 × 10-2) NS 1.3 (2.67 × 10-3) 2.7 (9.54 × 10-4)

-2.4 (7.29 × 10 ) 2.4 (7.77 × 10-3) 1.6 (3.26 × 10-3) NS 3.0 (2.51 × 10-3) NS 6.7 (1.89 × 10-4)

-5.2 (1.88 × 10-3)

3.4 (1.66 × 10-3)

2.2 (1.08 × 10-3) 1.8 (7.86 × 10-5)

NS NS NS NS NS NS NS

-6.6 (1.07 × 10-4)

-1.6 (3.09 × 10-3)

-4

7.1 (2.92 × 10-4)

NS

NS

5.1 (5.33 × 10-4) -2.0 (5.79 × 10 )

NS

3.0 (2.98 × 10-3)

-3

-2.4 (7.71 × 10-4) -1.7 (2.07 × 10 )

-4.5 (1.74 × 10-6) -2

NS -2.4 (1.62 × 10-2)

NS 2.4 (8.25 × 10 )

-3

1.6 (2.42 × 10-2)

-4.7 (9.52 × 10-3) 2.0 (1.50 × 10-2) 2.4 (6.94 × 10-3) 3.2 (1.03 × 10-3)

-2.0 (1.63 × 10-3)

-1.4 (4.88 × 10-2) NS 2.3 (2.77 × 10-4) NS 1.4 (8.38 × 10-3)

-2.1 (6.00 × 10-4)

NS -2.8 (1.47 × 10-3) 2.3 (1.66 × 10-4)

-2.3 (3.35 × 10-3) -2.6 (1.91 × 10-3) 1.9 (3.60 × 10-3) NS

-2.7 (1.08 × 10-4)

-1.5 (4.97 × 10-3)

2.1 (1.43 × 10-2)

2.2 (2.90 × 10 )

NS

NS

NS

NS

NS

1.6 (2.89 × 10-3)

NS NS NS NS

NS

NS

NS NS NS

NS

unique DSM 1731/ peptides peptides Rh8 solventogenesis/ Rh8 squence matched/ detected MASCOT acidogenesis/DSM DSM 1731 solventogenesis/ e f gene coverage (%) searched by MS/MS score 1731 acidogenesis acidogenesis Rh8 acidogenesis

Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant

research articles

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

3056

Journal of Proteome Research • Vol. 9, No. 6, 2010 gi|15023348 gi|15023180 gi|15024092

gi|15023135 gi|15025287 gi|15022975

gi|15026393 gi|15023435 gi|15026727

gi|15024502 gi|15026150

gi|15026284

gi|15022823

gi|15024657 gi|15025108 gi|15024224 gi|15025220 gi|15023463

gi|15025199

gi|15023156 gi|60391227 gi|15026203 gi|15026268 gi|15024226

gi|15025667

NCBI accession number

13506/5.05 19463/5.09 20978/5.54

34721/6.51 43200/7.2 24596/8.99

22826/5.70 24206/5.50 25494/8.04

19323/6.64 54539/4.76

55731/4.92

71570/5.80

39365/4.87 24056/4.57 33833/9.05 38121/8.86 31812/5.50

29503/5.78

24964/5.35 27284/5.14 43425/5.04 17701/4.70 21921/4.92

21410/5.03

theor. Mrc/ pId

CAC0488 CAC0334 CAC1171

CAC0293 CAC2282 tgt CAC0148

CAC3314 CAC0567 CAC3627

CAC1551 CAC3086

CAC3212

CAC0006 gyrB

CAC1693 ftsZ CAC2118 CAC1295 CAC2222 cheB CAC0594 pdxY

CAC2203 hag

CAC0313 CAC3111 map CAC3136 tufA CAC3198 greA CAC1297

41 39 54

33 38 43

67 36 31

52 25

48

24

15/20

1 3 3

2 3 4

4 4 3

1 1

111 170 165

119 87 257

217 243 61

144 106

110

99

3

662 296 60 176 633

467

356 427 135 312 106

101

6 6 1 3 5

3

Others 58 77 70 36 46 73

3 5 3 5 2

3

72 74 36 82 26

32

-2.6 (5.24 × 10-3)

-1.6 (9.11 × 10-3)

NS 2.7 (2.01 × 10-3) 1.7 (2.59 × 10-2)

2.7 (1.15 × 10-3) NS NS

NS NS 3.2 (1.39 × 10-2)

NS 8.7 (1.25 × 10-2) 10.9 (2.36 × 10-3)

-2.2 (3.71 × 10-3) 3.9 (2.08 × 10-3) 7.1 (5.75 × 10-4)

NS NS NS NS NS NS

NS NS 2.7 (3.47 × 10-4) 3.9 (1.07 × 10-6) NS

NS NDh

4.7 (4.90 × 10-4) NDh

NS

NS

NS 2.0 (1.47 × 10-2) 4.4 (3.69 × 10-3) 2.7 (1.80 × 10-3) 1.2 (4.78 × 10-2)

1.7 (1.49 × 10-2)

1.4 (6.55 × 10-3) 1.6 (1.14 × 10-4) 2.2 (8.71 × 10-3) 2.6 (6.91 × 10-4) 3.6 (1.64 × 10-3)

2.6 (5.37 × 10-3)

NS NS 2.9 (1.46 × 10-3)

5.8 (3.71 × 10 ) NS

-4

-4.3 (2.97 × 10 ) NS NS NS -4.9 (2.15 × 10-3)

-4

-3.5 (3.04 × 10-4)

NS 1.3 (1.10 × 10-2) 2.3 (4.18 × 10-4) NS NS

NS

NS NS NS NS -2.7 (6.68 × 10-5)

-12.3 (4.47 × 10-5)

NS NS NS NS NS

NS

unique DSM 1731/ peptides peptides Rh8 solventogenesis/ Rh8 squence matched/ detected MASCOT acidogenesis/DSM DSM 1731 solventogenesis/ e f gene coverage (%) searched by MS/MS score 1731 acidogenesis acidogenesis Rh8 acidogenesis

CAC2640 clpP

gene locus

ratiog (P-value)

a Protein identification numbers (Spots ID) correspond to the numbers in Figure S3 of Supporting Information 2. b The protein description was primarily based on the genome annotation of C. acetobutylicum ATCC 824. c theor. Mr is abbreviation of theoretical molecular mass. d theor. pI is abbreviation of theoretical isoelectric point. e Protein identified by PMF. The number of peptides that match/searched peptides was provided. f Protein identified by peptide mass fingerprint (PMF) and MS/MS. -: No unique peptides detected by MS/MS. g Differential protein expression (ratio) of corresponding protein. NS: No significant change. h Protein spot not detected in the strain DSM 1731.

100 101 102

97 98 99

94 95 96

92 93

91

90

85 86 87 88 89

84

Possible hook-associated protein, flagellin family Cell division GTPase FtsZ Cell division protein DivIVA ERA GTPase Chemotaxis protein CheB Predicted phosphate-utilizing enzyme involved in pyridoxine/ purine/histidine biosynthesis DNA gyrase (topoisomerase II) B subunit Fusion of Uroporphyrinogen-III methylase related protein and MAZG family protein, YABN B.subtilis Nitroreductase family protein Protein containing cell adhesion domain Nitroreductase family protein Putative methyltransferase PP-loop superfamily ATPase, confers aluminumresistance MccF-like protein Queuine tRNA-ribosyltransferase Predicted enzyme with TIM-barrel fold hypothetical protein CAC0488 Hypothetical protein, CF-21 family Hypothetical protein CAC1171

Protease subunits of ATP-dependent protease, ClpP Phage shock protein A Methionine aminopeptidase Elongation Factor Tu (Ef-Tu) Transcription elongation factor, greA N-Terminal fragment of elongation factor Ts

78

79 80 81 82 83

protein descriptionb

match ida

Table 1. Continued

research articles Mao et al.

Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant

research articles

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

the hierarchical clustering analysis and heat map visualization were performed with TIGR MeV.43 The hierarchical clustering analysis allowed the discrimination of four distinct profiles of protein expression (Figure 5). Cluster 1 contained 38 proteins upregulated in solventogenesis, whereas the protein expression levels in mutant Rh8 were generally higher than that of strain DSM 1731 in both acidogenesis and solventogenesis. The majority of proteins (17/38) grouped in this cluster are involved in solvent formation, protein folding, cellular regulation, and cell division (Figure 5). Cluster 2 contained 15 proteins that were generally stable between acidogenic and solventogenic phases but significantly upregulated in mutant Rh8. Seven out of 15 are involved in solvent formation, protein folding, and transport. Cluster 3 contained 36 proteins downregulated in solventogenesis, whereas the protein expression levels in mutant Rh8 were generally lower than that of strain DSM 1731 in both acidogenesis and solventogenesis. Interestingly, 19 out of 36 are involved in amino acid metabolism and protein synthesis. Cluster 4 contained 13 proteins slightly upregulated

in solventogenesis, whereas the protein expression levels of strain Rh8 was generally lower than that of DSM 1731 in solventogenesis. Proteins grouped in this cluster were mainly involved in nucleotide metabolism and transport. Transcriptional Analysis of Selected Genes by Real Time RT-PCR. To investigate whether the proteins showing altered levels on 2-DE are in good accordance with the changes at the transcriptional level, we selected 11 genes whose encoded proteins were found differentially regulated on 2-DE and the mRNA transcript levels were measured by using real time RTPCR. Interestingly, transcriptional regulation of all selected genes showed a positive correlation with the proteomic patterns of the identified proteins. Genes corresponding to the proteins that were up-regulated in proteomic studies, such as ilvB, ctfA, ctfB, groEL, hsp18, dnaK, natA, bdhB, adhE1, and thl, were also up-regulated at the mRNA level (Figure 6). Hag that was down-regulated at the proteome level was also downregulated at the mRNA level (hag) (Figure 6). Journal of Proteome Research • Vol. 9, No. 6, 2010 3057

research articles

Mao et al. Solvent-tolerant strains were obtained through serial enrichment of liquid cultures with butanol. Strains SA-113 and G119 were developed in this manner and were tolerant to 15 and 18 g/L butanol. Butanol tolerant mutant C. acetobutylicum SA-2 derived from strain ATCC 824 by classic chemical mutagenesis could grow in the presence of 18 g/L butanol.14 However, the SA-2 strain could only produce trace amount of butanol,14 suggesting the improved tolerance did not result in a proportional increase in butanol production. A C. beijerinckii strain BA101 generated by chemical mutagenesis has been shown to produce 19 g/L butanol.61 Overexpression of spo0A also conferred increased tolerance and prolonged metabolism in response to butanol stress.55 In addition, overexpression of groESL in C. acetobutylicum has been shown to confer the host increased butanol tolerance and 17.1 g/L butanol was produced.15 Moreover, the buk gene inactivated ATCC 824 strain could produce 16.7 g/L butanol.16 Furthermore, inactivation of solR gene, combined with increased adhE1 (previously designated as aad) gene expression in ATCC 824 strain, resulted in a strain with improved butanol production (17.6 g/L).17 The butanol-tolerant mutant Rh8 that we developed after extensive mutagenesis and genome shuffling is able to tolerate 19 g/L butanol and can produce 15.3 g/L butanol. Although the butanol titer produced by strain Rh8 is not the highest among the literatures, we were able to perform comparative proteomic analysis between the mutant strain and the base strain of C. acetobutylicum, for which very limited proteomic data have been previously reported.

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

Discussion A high resolution 2-DE proteome reference map of C. acetobutylicum is essential for comparative proteomic analyses to investigate the difference among different growth stages, or among the wild type and the various mutants. Previous proteomic analysis of C. acetobutylicum mainly focused on acidic proteins6,7 here we cover both acidic and basic proteins. We identified 564 proteins account for 14.7% of the predicted 3848 ORFs in the genome. This percentage is in a good accordance with the recently published proteome reference maps of other microorganisms, where the percentage of the identification proteins is usually between 5-21.3% (e.g., 5.9% for Corynebacterium efficiens YS-314,57 4.6% for Corynebacterium glutamicum ATCC 14067,58 5.7% for Bacillus anthracis A16R,59 12.8% for Neisseria meningitidis serogroup A,60 and 21.3% for Bifidobacterium longum NCC270550). The identified proteins were used to reconstruct the metabolic network of C. acetobutylicum DSM 1731 to evaluate the quality and the coverage of the proteome reference map. More than 50% of the proteins involved in major metabolic pathways are present in the reference proteome map. The wide coverage of the identified proteins in the metabolic pathways indicates a good application potential of using this proteome reference map to understand the metabolism-related physiology of C. acetobutylicum. 3058

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

Comparative proteomic analysis between the wild type strain DSM 1731 and the mutant Rh8 in acidogenesis and solventogenesis revealed a total of 102 differentially expressed proteins. The mechanism that strain Rh8 developed for an improved butanol tolerance can be seen from two aspects: upregulate proteins (e.g., chaperones and solvent formation related) in acidogenic phase, and further upregulate these proteins in solventogenic phase; or downregulate proteins (e.g., amino acid metabolism and protein synthesis related) in acidogenic phase, and further downregulate these proteins in solventogenic phase. This suggests that strain Rh8 may have developed a mechanism to prepare itself for coping with butanol challenges before butanol is produced, leading to an increased butanol production. It has been previously reported that chaperone proteins play an important role in increasing solvent production and tolerance of C. acetobutylicum.15 The well-characterized chaperone proteins 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 102 differentially expression proteins identified in this study were chaperone proteins (Table 1). The 16 upregulated chaperone proteins in Rh8 can be categorized into three groups: only upregulated in acidogenic phase (HtpG, GroEL, DnaK, and HtrA); or only in solventogenic phase (YacI, GroES, ClpP, Map, TufA, GreA, CAC1297, CAC0313, and CAC3598); or in both acidogenic and solventogenic phase (Hsp18, GrpE, and ClpC). This resulted in a comparable or even higher total upregulation of chaperone proteins in strain Rh8 as compared with strain DSM 1731, which might contribute to the increased butanol tolerance of strain Rh8. These results suggest that increased production of chaperone proteins might be a general consequence in C. acetobutylicum that can be either induced by butanol stress20 or by adaptation to a high butanol concentration as shown in this study.

Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant Interestingly, 5 out of the 16 chaperone proteins (TufA, GreA, CAC1297, CAC0313, CAC3598) were found for the first time in C. acetobutylicum that might contribute to the increased butanol tolerance. Within these 5 proteins, three elongation factors, such as TufA, GreA, and N-terminal fragment of elongation factor Ts (Tsf) seem to be involved in protein-protein interaction with unfolded proteins.62 TufA interacts with unfolded and denatured proteins as do molecular chaperones after stress.62 In Streptococcus mutans, an increase expression of TufA was found during acid-tolerant growth stage.63 GreA is one of the only few proteins that are upregulated during lowpH growth of Streptococcus mutans,63 and one of the only 9 proteins that are up-regulated in Staphylococcus aureus in response to a challenge by cell wall-active antibiotic.64 In E. coli, overexpression of greA confers resistance to toxic levels of divalent metal ions such as Zn2+ and Mn2+.65 Finally, Tsf has been reported as a steric chaperone by functioning as a structural template for the correct folding of TufA.66 In E. coli, the protein denaturant guanidine hydrochloride stress-induced increased expression of Tsf.67 These findings suggest that elongation factors may play important roles in butanol tolerance. Ten out of the 102 differentially expression proteins in this study are proteins involved in solvent formation (Table 1), which are known to increase expression at the onset of solventogenesis.21 The interesting observation is that 7 out of 10 proteins (THL, AdhE1, CtfA/B, Adc, BdhA/B) in strain Rh8 that are involved in acetone and butanol production significantly upregulated in acidogenic phase, resulting in a higher total expression level in solventogenesis as compared to strain DSM 1731. We believe the significant upregulation of these solvent formation proteins contributed to the increased production of acetone and butanol in mutant Rh8. C. acetobutylicum strain ATCC 824 with GroESL when challenged with 0.75%(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 solventogenic operon adhE1-ctfA-ctfB.12 This suggests that solvent formation proteins are not only regulated by short-term butanol stress, but can also be regulated by long-term adaptation to a higher butanol concentration. Twenty-five proteins out of 102 differentially expression proteins are involved in amino acid metabolism and protein synthesis (Table 1), which represents the largest functional group. The majority of these proteins were grouped in Cluster 3 of Figure 5, showing that Rh8 might develop a mechanism to slow down amino acid metabolism and protein synthesis to adapt to butanol challenge. Previous studies have indicated that amino acid metabolism and protein synthesis is affected by butanol stress, but the data were quite difficult to interpret some pathways were upregulated, whereas some downregulated.12 Our study demonstrated that proteins involved in amino acid metabolism and protein synthesis of Rh8 showed a general trend of downregulation in both acidogenic and solventogenic phases. The decreased amino acid metabolism and protein synthesis could also be reflected by the reduced specific growth rate and the reduced maximum optical density of strain Rh8 in solventogenesis (Figure 4B). In addition, of the 11 proteins involved in amino acid metabolism, glutamine synthetase type III (GlnA, CAC2658) and glycine hydroxymethyltransferase (GlyA, CAC2264) were found for the first time to respond to the shift from acidogenic to solventogenic phase (Table 1).

research articles

The expression of 7 aminoacyl-tRNA synthetases downregulated, which was quite consistent with a previous DNA microarray study showing half aminoacyl-tRNA synthetases downregulated when entering the solventogenic phase.21 One exception is SerS, which was downregulated in the present study but upregulated in C. acetobutylicum ATCC 82421 and DSM 7926 at solventogenic phase. In fact, the serC-serA-serHTHserS (CAC0014-CAC0017) operon was downregulated by 1.5to 4.1-fold in strains DSM 1731 and Rh8 at solventogenic phase (Table 1), while this operon was upregulated in ATCC 824. Currently, it remains unclear why aminoacyl-tRNA synthetases show a trend of downregulation in solventogenic phases.21 We identified a ribosome-associated protein Y (CAC2847) that was upregulated in solventogenic phase in both strain DSM 1731 and mutant Rh8. This protein inhibits translation by interfering with the binding of the aminoacyl-tRNA to the ribosomal A site, resulting in an inhibited protein synthesis at the elongation stage of translation.68 The decreased need for aminoacyl-tRNA might trigger downregulation of 7 aminoacyl-tRNA synthetases. We postulate that the decreased amino acid metabolism and protein synthesis may save energy for strain Rh8, therefore improving the energy utilization efficiency of strain Rh8 which might be a key for increasing butanol tolerance.

Conclusions Proteomics tool offers new opportunities to observe global cellular events by directly visualizing a large set of gene expression products. In this study, 2-DE and MS were employed to establish the proteome reference map of C. acetobutylicum. This represents the most extensive investigation of the C. acetobutylicum proteome to date, providing general and fundamental proteomic data for the Clostridium community. The availability of the proteome reference map allowed us to better understand mutants with different phenotypes and discovery of novel butanol tolerance mechanism. Comparative proteomic analysis between the wild type DSM 1731 and the butanol tolerant mutant Rh8 in acidogenic/solventogenic phases revealed a total of 102 differentially expressed proteins that are mainly involved in protein folding, solvent formation, amino acid metabolism, protein synthesis, nucleotide metabolism, transport, and others. Hierarchical clustering analysis of 102 differentially expressed proteins revealed that Rh8 cells might have developed a mechanism to prepare themselves well-ready for butanol challenge before butanol is produced, leading to an increased butanol yield. Further studies on the mechanisms of butanol tolerance will allow identification of essential butanol stress- and hyperproductivity-related genes, clarification of the molecular basis of adaptive response and cross protection, and strategies to select or improve strains for efficient butanol production. Abbreviations: 2-DE, two-dimensional gel electrophoresis; CAI, codon adaptation index; CHAPS, 3-[(3-cholamidopropyl)dimethylamonio]-1-propanesulfonate; CHCA, a-cyano-4-hydroxycinnamic acid; GRAVY, grand average of hydrophobicity; PRPP, phosphoribosyl pyrophosphate.

Acknowledgment. This work was supported the National High Technology Research and Development Program of China (863 Project, No.2006AA02Z237), National Key Fundamental Research and Development Project of China (973 Project, No.2007CB707803), Knowledge Innovation Program of CAS (No. KSCXZ-YW-G-007), and Hundreds Talents Program of the Chinese Academy of Sciences. We thank Jie Zhou for helpful discussions and suggestions. Journal of Proteome Research • Vol. 9, No. 6, 2010 3059

research articles Supporting Information Available: Supplemental figures, tables, and PMF. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Ezeji, T.; Qureshi, N.; Blaschek, H. P. Butanol production from agricultural residues: Impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. Biotechnol. Bioeng. 2007, 97 (6), 1460–1469. (2) Qureshi, N.; Blaschek, H. P. ABE production from corn: a recent economic evaluation. J. Ind. Microbiol. Biotechnol. 2001, 27 (5), 292–297. (3) Lee, S. Y.; Park, J. H.; Jang, S. H.; Nielsen, L. K.; Kim, J.; Jung, K. S. Fermentative butanol production by Clostridia. Biotechnol. Bioeng. 2008, 101 (2), 209–228. (4) Tummala, S. B.; Tomas, C.; Harris, L. M.; Welker, N. E.; Rudolph, F. B.; Bennett, G. N.; Papoutsakis, E. T. Genetic tools for solventogenic Clostridia. In Clostridia: Biotechnology and medical applications; John Wiley & Sons: New York, 2001; pp 105-123. (5) Nolling, J.; Breton, G.; Omelchenko, M.; Makarova, K.; Zeng, Q.; Gibson, R.; Lee, H.; Dubois, J.; Qiu, D.; Hitti, J. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J. Bacteriol. 2001, 183 (16), 4823–4838. (6) Schaffer, S.; Isci, N.; Zickner, B.; Durre, P. Changes in protein synthesis and identification of proteins specifically induced during solventogenesis in Clostridium acetobutylicum. Electrophoresis 2002, 23 (1), 110–121. (7) Sullivan, L.; Bennett, G. N. Proteome analysis and comparison of Clostridium acetobutylicum ATCC 824 and Spo0A strain variants. J. Ind. Microbiol. Biotechnol. 2006, 33 (4), 298–308. (8) Jones, D. T.; Woods, D. R. Acetone-butanol fermentation revisited. Microbiol. Rev. 1986, 50 (4), 484–524. (9) Bowles, L. K.; Ellefson, W. L. Effects of butanol on Clostridium acetobutylicum. Appl. Environ. Microbiol. 1985, 50 (5), 1165–1170. (10) Vollherbst-Schneck, K.; Sands, J. A.; Montenecourt, B. S. Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 1984, 47 (1), 193– 194. (11) Terracciano, J. S.; Kashket, E. R. Intracellular conditions required for Initiation of solvent production by Clostridium acetobutylicum. Appl. Environ. Microbiol. 1986, 52 (1), 86–91. (12) Alsaker, K. V.; Paredes, C.; Papoutsakis, E. T. Metabolite stress and tolerance in the production of biofuels and chemicals: Geneexpression-based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum. Biotechnol. Bioeng. 2009, 105 (6), 1131–1147. (13) Lin, Y. L.; Blaschek, H. P. Butanol production by a butanol-tolerant strain of Clostridium acetobutylicum in extruded corn broth. Appl. Environ. Microbiol. 1983, 45 (3), 966–973. (14) Baer, S. H.; Blaschek, H. P.; Smith, T. L. Effect of butanol challenge and temperature on lipid composition and membrane fluidity of butanol-tolerant Clostridium acetobutylicum. Appl. Environ. Microbiol. 1987, 53 (12), 2854–2861. (15) Tomas, C. A.; Welker, N. E.; Papoutsakis, E. T. Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell’s transcriptional program. Appl. Environ. Microbiol. 2003, 69 (8), 4951–4965. (16) Harris, L. M.; Desai, R. P.; Welker, N. E.; Papoutsakis, E. T. Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition. Biotechnol. Bioeng. 2000, 67 (1), 1–11. (17) Harris, L. M.; Blank, L.; Desai, R. P.; Welker, N. E.; Papoutsakis, E. T. Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene. J. Ind. Microbiol. Biotechnol. 2001, 27 (5), 322–328. (18) Harris, L. M.; Welker, N. E.; Papoutsakis, E. T. Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824. J. Bacteriol. 2002, 184 (13), 3586–3597. (19) Soucaille, P.; Joliff, G.; Izard, A.; Goma, G. Butanol tolerance and autobacteriocin production by Clostridium acetobutylicum. Curr. Microbiol. 1987, 14 (5), 295–299. (20) Tomas, C. A.; Beamish, J.; Papoutsakis, E. T. Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum. J. Bacteriol. 2004, 186 (7), 2006–2018. (21) Alsaker, K. V.; Papoutsakis, E. T. Transcriptional program of early sporulation and stationary-phase events in Clostridium acetobutylicum. J. Bacteriol. 2005, 187 (20), 7103–7118.

3060

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

Mao et al. (22) Tomas, C. A.; Alsaker, K. V.; Bonarius, H. P.; Hendriksen, W. T.; Yang, H.; Beamish, J. A.; Paredes, C. J.; Papoutsakis, E. T. DNA array-based transcriptional analysis of asporogenous, nonsolventogenic Clostridium acetobutylicum strains SKO1 and M5. J. Bacteriol. 2003, 185 (15), 4539–4547. (23) Guillot, A.; Gitton, C.; Anglade, P.; Mistou, M. Y. Proteomic analysis of Lactococcus lactis, a lactic acid bacterium. Proteomics 2003, 3 (3), 337–354. (24) Drews, O.; Reil, G.; Parlar, H.; Gorg, A. Setting up standards and a reference map for the alkaline proteome of the Gram-positive bacterium Lactococcus lactis. Proteomics 2004, 4 (5), 1293–1304. (25) Anglade, P.; Demey, E.; Labas, V.; Le Caer, J. P.; Chich, J. F. Towards a proteomic map of Lactococcus lactis NCDO 763. Electrophoresis 2000, 21 (12), 2546–2549. (26) O’Farrell, P. H. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 1975, 250 (10), 4007–4021. (27) Hermann, T.; Pfefferle, W.; Baumann, C.; Busker, E.; Schaffer, S.; Bott, M.; Sahm, H.; Dusch, N.; Kalinowski, J.; Puhler, A.; Bendt, A. K.; Kramer, R.; Burkovski, A. Proteome analysis of Corynebacterium glutamicum. Electrophoresis 2001, 22 (9), 1712–1723. (28) Zhao, B.; Yeo, C. C.; Poh, C. L. Proteome investigation of the global regulatory role of sigma 54 in response to gentisate induction in Pseudomonas alcaligenes NCIMB 9867. Proteomics 2005, 5 (7), 1868–1876. (29) Antelmann, H.; Williams, R. C.; Miethke, M.; Wipat, A.; Albrecht, D.; Harwood, C. R.; Hecker, M. The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C12. Proteomics 2005, 5 (14), 3684–3695. (30) Hirsch, A.; Grinsted, E. Methods for the growth and enumeration of anaerobic spore-formers from cheese, with observations on the effect of nisin. J. Dairy Res. 1954, 21, 101–110. (31) Klijn, N.; Nieuwenhof, F. F. J.; Hollwerf, J. D.; Vanderwaals, C. B.; Weerkamp, A. H. Identification of Clostridium tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification. Appl. Environ. Microbiol. 1995, 61 (8), 2919– 2924. (32) Slade, S. J.; Harris, R. F.; Smith, C. S.; Andrews, J. H.; Nordheim, E. V. Microplate assay for Colletotrichum spore production. Appl. Environ. Microbiol. 1987, 53 (4), 627–632. (33) Wiesenborn, D. P.; Rudolph, F. B.; Papoutsakis, E. T. Thiolase from Clostridium acetobutylicum ATCC 824 and its role in the synthesis of acids and solvents. Appl. Environ. Microbiol. 1988, 54 (11), 2717– 2722. (34) Zhang, Y. X.; Perry, K.; Vinci, V. A.; Powell, K.; Stemmer, W. P.; del Cardayre, S. B. Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 2002, 415 (6872), 644–646. (35) Annous, B. A.; Blaschek, H. P. Regulation and localization of amylolytic enzymes in Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 1990, 56 (8), 2559–2561. (36) O’brien, R.; Morris, J. Oxygen and the growth and metabolism of Clostridium acetobutylicum. J. Gen. Microbiol. 1971, 68 (3), 307– 318. (37) Allcock, E. R.; Reid, S. J.; Jones, D. T.; Woods, D. R. Clostridium acetobutylicum protoplast formation and regeneration. Appl. Environ. Microbiol. 1982, 43 (3), 719–721. (38) Nair, R. V.; Bennett, G. N.; Papoutsakis, E. T. Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824. J. Bacteriol. 1994, 176 (3), 871– 885. (39) Wildgruber, R.; Reil, G.; Drews, O.; Parlar, H.; Gorg, A. Web-based two-dimensional database of Saccharomyces cerevisiae proteins using immobilized pH gradients from pH 6 to pH 12 and matrixassisted laser desorption/ionization-time of flight mass spectrometry. Proteomics 2002, 2 (6), 727–732. (40) Falsone, S. F.; Gesslbauer, B.; Rek, A.; Kungl, A. J. A proteomic approach towards the Hsp90-dependent ubiquitinylated proteome. Proteomics 2007, 7 (14), 2375–2383. (41) Fernandez-Arenas, E.; Cabezon, V.; Bermejo, C.; Arroyo, J.; Nombela, C.; Diez-Orejas, R.; Gil, C. Integrated proteomics and genomics strategies bring new insight into Candida albicans response upon macrophage interaction. Mol. Cell. Proteomics 2007, 6 (3), 460–478. (42) Chaze, T.; Meunier, B.; Chambon, C.; Jurie, C.; Picard, B. In vivo proteome dynamics during early bovine myogenesis. Proteomics 2008, 8 (20), 4236–4248. (43) Saeed, A. I.; Sharov, V.; White, J.; Li, J.; Liang, W.; Bhagabati, N.; Braisted, J.; Klapa, M.; Currier, T.; Thiagarajan, M.; Sturn, A.; Snuffin, M.; Rezantsev, A.; Popov, D.; Ryltsov, A.; Kostukovich, E.; Borisovsky, I.; Liu, Z.; Vinsavich, A.; Trush, V.; Quackenbush, J. TM4: a free, open-source system for microarray data management and analysis. BioTechniques 2003, 34 (2), 374–378.

research articles

Wild Type Clostridium acetobutylicum DSM 1731 and its Mutant (44) Peden, J. F. Analysis of codon usage. PhD Thesis, University of Nottingham, Nottingham, U.K., 1999. (45) Tatusov, R. L.; Koonin, E. V.; Lipman, D. J. A genomic perspective on protein families. Science 1997, 278 (5338), 631–637. (46) Hiller, K.; Schobert, M.; Hundertmark, C.; Jahn, D.; Munch, R. JVirGel: Calculation of virtual two-dimensional protein gels. Nucleic Acids Res. 2003, 31 (13), 3862–3865. (47) Behrens, M.; Schreiber, W.; Du ¨ rre, P. The high-affinity K+translocating ATPase complex from Clostridium acetobutylicum consists of six subunits. Anaerobe 2001, 7 (3), 159–169. (48) Link, A. J.; Robison, K.; Church, G. M. Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12. Electrophoresis 1997, 18 (8), 1259–1313. (49) Buttner, K.; Bernhardt, J.; Scharf, C.; Schmid, R.; Mader, U.; Eymann, C.; Antelmann, H.; Volker, A.; Volker, U.; Hecker, M. A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis. Electrophoresis 2001, 22 (14), 2908–2935. (50) Yuan, J.; Zhu, L.; Liu, X.; Li, T.; Zhang, Y.; Ying, T.; Wang, B.; Wang, J.; Dong, H.; Feng, E.; Li, Q.; Wang, H.; Wei, K.; Zhang, X.; Huang, C.; Huang, P.; Huang, L.; Zeng, M. A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705. Mol. Cell. Proteomics 2006, 5 (6), 1105–1118. (51) Kyte, J.; Doolittle, R. F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 1982, 157 (1), 105– 132. (52) Bahl, H.; Andersch, W.; Gottschalk, G. Continuous production of acetone and butanol by Clostridium acetobutylicum in a two-stage phosphate limited chemostat. Appl. Microbiol. Biotechnol. 1982, 15 (4), 201–205. (53) Walter, K. A.; Bennett, G. N.; Papoutsakis, E. T. Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes. J. Bacteriol. 1992, 174 (22), 7149–7158. (54) Welch, R. W.; Rudolph, F. B.; Papoutsakis, E. T. Purification and characterization of the NADH-dependent butanol dehydrogenase from Clostridium acetobutylicum (ATCC 824). Arch. Biochem. Biophys. 1989, 273 (2), 309–318. (55) Alsaker, K. V.; Spitzer, T. R.; Papoutsakis, E. T. Transcriptional analysis of spo0A overexpression in Clostridium acetobutylicum and its effect on the cell’s response to butanol stress. J. Bacteriol. 2004, 186 (7), 1959–1971. (56) Jones, S. W.; Paredes, C. J.; Tracy, B.; Cheng, N.; Sillers, R.; Senger, R. S.; Papoutsakis, E. T. The transcriptional program underlying the physiology of clostridial sporulation. Genome Biol. 2008, 9 (7), R114. (57) Hansmeier, N.; Chao, T. C.; Puhler, A.; Tauch, A.; Kalinowski, J. The cytosolic, cell surface and extracellular proteomes of the

(58)

(59)

(60)

(61)

(62)

(63)

(64)

(65)

(66)

(67)

(68)

biotechnologically important soil bacterium Corynebacterium efficiens YS-314 in comparison to those of Corynebacterium glutamicum ATCC 13032. Proteomics 2006, 6 (1), 233–250. Li, L.; Wada, M.; Yokota, A. Cytoplasmic proteome reference map for a glutamic acid-producing Corynebacterium glutamicum ATCC 14067. Proteomics 2007, 7 (23), 4317–4322. Wang, J. J.; Ying, T. Y.; Wang, H. L.; Shi, Z. X.; Li, M. Z.; He, K.; Feng, E. L.; Wang, J.; Yuan, J.; Li, T.; Wei, K. H.; Su, G. F.; Zhu, H. C.; Zhang, X. M.; Huang, P. T.; Huang, L. Y. 2-D reference map of Bacillus anthracis vaccine strain A16R proteins. Proteomics 2005, 5 (17), 4488–4495. Bernardini, G.; Renzone, G.; Comanducci, M.; Mini, R.; Arena, S.; D’Ambrosio, C.; Bambini, S.; Trabalzini, L.; Grandi, G.; Martelli, P.; Achtman, M.; Scaloni, A.; Ratti, G.; Santucci, A. Proteome analysis of Neisseria meningitidis serogroup A. Proteomics 2004, 4 (10), 2893–2926. Formanek, J.; Mackie, R.; Blaschek, H. P. Enhanced butanol production by Clostridium beijerinckii BA101 grown in semidefined P2 medium containing 6% maltodextrin or glucose. Appl. Environ. Microbiol. 1997, 63 (6), 2306–2310. Caldas, T. D.; El Yaagoubi, A.; Richarme, G. Chaperone properties of bacterial elongation factor EF-Tu. J. Biol. Chem. 1998, 273 (19), 11478–11482. Len, A. C.; Harty, D. W.; Jacques, N. A. Stress-responsive proteins are upregulated in Streptococcus mutans during acid tolerance. Microbiology 2004, 150 (Pt 5), 1339–1351. Singh, V. K.; Jayaswal, R. K.; Wilkinson, B. J. Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach. FEMS Microbiol. Lett. 2001, 199 (1), 79–84. Susa, M.; Kubori, T.; Shimamoto, N. A pathway branching in transcription initiation in Escherichia coli. Mol. Microbiol. 2006, 59 (6), 1807–1817. Krab, I. M.; te Biesebeke, R.; Bernardi, A.; Parmeggiani, A. Elongation factor Ts can act as a steric chaperone by increasing the solubility of nucleotide binding-impaired elongation factor-Tu. Biochemistry 2001, 40 (29), 8531–8535. Han, K. Y.; Song, J. A.; Ahn, K. Y.; Park, J. S.; Seo, H. S.; Lee, J. Enhanced solubility of heterologous proteins by fusion expression using stress-induced Escherichia coli protein, Tsf. FEMS Microbiol. Lett. 2007, 274 (1), 132–138. Agafonov, D. E.; Kolb, V. A.; Spirin, A. S. Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage. EMBO Rep. 2001, 2 (5), 399–402.

PR9012078

Journal of Proteome Research • Vol. 9, No. 6, 2010 3061