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Supporting Information for Proteomics DOI 10.1002/Pmic.200600987 Supporting Information for Proteomics DOI 10.1002/pmic.200600987 Lars Whlbrand, Birte Kallerhoff, Daniela Lange, Peter Hufnagel, Jrgen Thiermann, Richard Reinhardt and Ralf Rabus Functional proteomic view of metabolic regulation in “Aromatoleum aromaticum” strain EbN1 ª 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Content 1. General proteome features Page Experimental outline of the presented study with “Aromatoleum aromaticum” 1-2 strain EbN1 (Fig. 1.1.) Spot pattern comparison of differently visualized 2DE gels (Fig. 1.2.) 1-3 Principle component analysis of the proteins for each analyzed 2D DIGE gel 1-4 of all main physiological groups (Fig. 1.3.) Fold changes in protein abundance of all identified proteins (Fig 1-5 Number of regulated as well as not regulated protein spots on the 2DE gels 1-6 and their share of total protein (Table 1.1.) Mean average ratio of not regulated protein spots of the three main 1-7 physiological groups (Table 1.2.) Mean average ratios of not regulated proteins, identified in all three main 1-8 physiological groups (Table 1.3.) Proteins identified in this study (Table 1.4.) 1-9 2. Cellular functions* 2.1. DNA replication, recombination and repair 2-2 2.1.1. DNA replication 2-2 2.2. Transcription 2-4 2.2.1. DNA-dependent RNA polymerase and sigma factors 2-5 2.2.2. Factors affecting RNA polymerase 2-5 2.2.3. Classes of transcriptional regulators 2-5 2.3. Translation 2-6 2.3.1. tRNA synthesis 2-6 2.3.2. tRNA/rRNA modification 2-7 2.3.3. Translation factors 2-8 2.3.4. Ribosomal proteins and modifying factors 2-10 2.3.5. Heat shock proteins and chaperons 2-11 2.3.6. Other translational functions 2-14 2.3.7. Proteases 2-15 2.4. Cell envelope biosynthesis 2-16 2.4.1. N-acteylglucosamine and murein synthesis and turnover 2-16 2.4.2. Lipopolysaccharide synthesis 2-17 2.4.3. Porins and outer membrane proteins 2-17 2.5. Cell division 2-18 2.5.1. Chromosome partitioning 2-18 2.5.2. Cell division 2-19 2.6. Chemotaxis, motility and secretion 2-20 2.6.2. Twitching motility 2-20 2.6.3. sec-dependent pathway 2-20 2.7. Transport 2-21 2.8. Coenzymes and vitamins 2-22 3. Special degradation pathways* 3.1. Anaerobic aromatic compound degradation 3-2 3.1.1. Phenylalanine 3-2 3.1.2. Phenylacetate 3-5 3.1.3. Benzyl alcohol and benzaldehyde 3-8 3.1.3.1. Benzyl alcohol 3-8 3.1.3.2. Benzaldehyde 3-11 3.1.3.3. Regulation of the benzyl alcohol and benzaldehyde 3-12 pathway 3.1.3.4. Induction of mandelate-racemase during growth with 3-14 benzyl alcohol and benzaldehyde 3.1.4. p-Cresol 3-16 3.1.5. Phenol 3-19 3.1.6. p-Hydroxybenzoate- 3-21 3.1.6.1. Up-regulation of the proteins not related to the pathway 3-23 3.1.9. Benzoate 3-24 3.1.10. m-Hydroxybenzoate 3-26 3.1.11 o-Aminobenzoate 3-29 3.2. Aerobic aromatic compound degradation 3-31 3.2.1. Benzoate 3-31 3.2.2. Gentisate (2,5-Dihydroxybenzoate) 3-34 3.2.3. Phenylacetate 3-36 3.2.4. Phenylalanine 3-39 3.2.5. m-Hydroxybenzoate 3-42 3.3. Anaerobic degradation of aliphatic alcohols, ketones and organic acids 3-45 3.3.1. Alcohols and ketones 3-45 3.3.1.1. 2-Propanol 3-45 3.3.1.2. Acetone 3-48 3.3.1.3. 2-Butanol 3-50 3.3.1.4. 2-Butanone 3-52 3.3.2. Organic acids 3-54 3.3.2.1. Propionate 3-54 4. Central and other metabolic features* 4.4. Glycolysis and gluconeogenesis, glyoxylate shunt and TCA-cycle 4-2 4.4.1. Glycolysis and gluconeogenesis 4-2 4.4.2. Glyoxylate shunt 4-4 4.4.3. TCA-cycle 4-6 4.5. Respiratory energy generation 4-8 4.5.2. NADH:ubiquinol oxidoreductase 4-9 4.5.3. Oxygen respiration 4-9 4.5.5. Other related proteins 4-10 4.6. β-Oxidation 4-11 4.6.1. CoA-ligases 4-11 4.6.4. Acyl-CoA dehydrogenases 4-11 4.6.5. Electron transfer flavoproteins 4-12 4.6.6. Enoyl-CoA hydratases / isomerases 4-12 4.6.7. Short chain alcohol dehydrogenases 4-13 4.6.8. Thiolases 4-13 4.6.9. Other alcohol and aldehyde dehydrogenases 4-13 4.7. Metalloenzymes and related proteins 4-15 4.7.7. Acoitase and endonuclease III 4-15 4.7.8. Ferredoxins 4-16 4.7.10 Fe-S cluster biosynthesis 4-16 4.7.12. Cytochrome c biosynthesis 4-17 4.7.15 Oxygen and NO detoxification 4-19 4.7.16. Peroxidases 4-20 4.7.18. Gene clusters related to iron transport 4-21 4.7.19. Transport of other metals 4-23 4.7.20. Zinc containing proteins 4-23 4.7.23 flavin enzymes 4-24 4.10. Hypothetical and conserved hypothetical proteins 4-25 5. Biosynthesis* 5.1. Amino acid synthesis 5-2 5.1.1. α-Ketoglutarate family 5-2 5.1.2. Aspartate family 5-4 5.1.3. Pyruvate family 5-7 5.1.4. Trioase family 5-9 5.1.5. Aromatic amino acid family 5-11 5.1.6. Histidine 5-13 5.3. Purine and pyrimidine synthesis 5-15 5.4. Lipid synthesis 5-17 5.6. Riboflavin synthesis 5-19 5.7. Folate synthesis 5-21 5.9. Panthothenic acid / CoA synthesis 5-22 5.15. Terpenid synthesis 5-23 5.16. Polyhydroxybutyrate synthesis and degradation 5-24 6. References * Numbering as used in supplementary material for genome sequence of strain EbN1 (Rabus et al., 2005; http://www.micro-genomes.mpg.de/ebn1/) 1. General proteome features 2DE (silver staining) Protein extraction Extract control and control Extract Extract control and control Extract qualitative protein profiling protein qualitative qualitative protein profiling protein qualitative Pal Pac BzOH BzalpCr Pol pHB mHB oAB Bz Bz GenPal Pac mHB Ac PrOH ActBuOH Bun Prp Ac Cultivation * -O -O -O -O -O -O -O -O -O -O +O2 +O2 +O2 O2 +O2 +O2 -O -O -O -O -O -O (> 5 passages) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Protein extraction Labellin Cy2 Cy3 Cy5 Cy2 Cy3 Cy5 Cy2 Cy3 Cy5 2DE (DIGE) quanttative protein profiling protein quanttative quanttative protein profiling protein quanttative 45 gels / 135 images 25 gels / 75 images 25 gels / 75 images to select protein spots for for identification spots protein to select to select protein spots for for identification spots protein to select Image analysis DeCyder DeCyder DeCyder 2DE (Coomassie) 93 gels / 4524 excised spots Protein identification Peptide mass fingerprinting by mass spectrometry m/z 354 identified proteins m/z (199 regulated vs. 155 non-regulated) Protein identification Protein Protein identification Protein Genome based 1 2 1 11 4 1 3 6 3 2 2 5 1 8 10 1 4 5 2 1 1 metabolic reconstruction Pal pCr mHB mHB/ Prp Bz Pol Pac Prp PrOH/Act/BuOH/Bun Bz Pac Pac Pal Prp Pal Gen Prp pCr Pal Bzal BzOH 250k Fig. 1.1. Experimental outline of the presented study with “Aromatoleum aromaticum” strain EbN1. Prior to proteomic analysis, cells were adapted for at least 5 passages to aerobic (blue) or anaerobic (red and orange) growth with the respective substrate. Names of indicated substrates are as follows: Pal, phenylalanine; Pac, phenylacetate; BzOH, benzyl alcohol; Bzal, benzaldehyde; pCr, p-cresol; Pol, phenol; pHB, p-hydroxybenzoate; mHB, m-hydroxybenzoate; oAB, o-aminobenzoate; Bz, benzoate; Gen, gentisate; Ac, acetate; PrOH, 2-propanol; Act, acetone; BuOH, 2-butanol; Bun, butanone, Prp; propionate. Quality control of the prepared protein extracts as well as qualitative protein profiling was done with silver-stained gels (top). Applying the 2D DIGE technique allowed quantitative protein profiling of independent protein extracts (center). The project was divided into three principle physiological groups: (i) anaerobic growth with aromatic compounds, (ii) aerobic growth with aromatic compounds and (iii) anaerobic growth with carboxylic acids, ketones and alcohols. Image analysis using the DeCyder software confirmed regulated spots as assigned by silver staining. In addition, more proteins could be identified as specifically regulated. Coomassie stained gels were used for protein identification (indicated). Selected regulated as well as non-regulated spots were excised and identified via mapping of peptide mass fingerprints. A B C D E F G H I Fig. 1.2. Spot pattern comparison of differently visualized 2DE gels. (A, D and G) silver stained gels, (B, E and H) false color images of 2D DIGE gels, (C, F and I) Coomassie Brilliant Blue staining. Independent protein extracts from cells adapted to anaerobic growth with m-hydroxybenzoate (red box), aerobic growth with phenylalanine (blue box) or anaerobic growth with 2-propanol (orange box) are shown exemplarily. 1-3 Proteins (Score Plot) Spot Maps (Loading Plot) 2.5 Benzaldehyd 0.3 Benzoat 2 Benzylalkoho 0.28 Phenol 1.5 0.26 Phenylacetat Phenylalanin 1 0.24 m-Hydroxyb o-Aminobenz 0.22 0.5 p-Cresol p-Hydroxybe 0.2 0 0.18 -0.5 0.16 -1 0.14 -1.5 0.12 PC2 PC2 0.1 -2 0.08 -2.5 0.06 -3 0.04 -3.5 0.02 -4 0 -0.02 -4.5 -0.04 -5 -0.06 A -5.5 -0.08 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 -0.05 0 0.05 0.1 0.15 PC1 PC1 Proteins (Score Plot) Spot Maps (Loading Plot) Acetat 1.8 0.38 Benzoat 1.6 0.36 Gentisat 1.4 0.34 Phenylacetat 1.2 0.32 Phenylalanin 1 0.3 m-Hydroxyb 0.8 0.28 0.6 0.26 0.4 0.24 0.2 0.22 0 0.2 -0.2 0.18 -0.4 0.16 -0.6 0.14 -0.8 0.12 -1 -1.2 0.1 PC2 -1.4 PC2 0.08 -1.6 0.06 -1.8 0.04 -2 0.02 -2.2 0 -2.4 -0.02 -2.6 -0.04 -2.8 -0.06 -3 -0.08 -3.2 -0.1 -3.4 -0.12 -3.6 -0.14 -3.8 -0.16 -4 -0.18 -4.2 B -0.2 -4.4 -0.22 -8 -6 -4 -2 0 2 4 6 -0.05 0 0.05 0.1 0.15 0.2 PC1 PC1 Proteins (Score Plot) Spot Maps (Loading Plot) 2.4 Acetat 0.38 Aceton 2.2 0.36 Butanol 2 0.34 Butanon 0.32 Propanol 1.8 0.3 Propionat 1.6 0.28 0.26 1.4 0.24 1.2 0.22 1 0.2 0.18 0.8 0.16 0.6 0.14 0.4 0.12 0.1 0.2 0.08 PC2 0 PC2 0.06 0.04 -0.2 0.02 -0.4 0 -0.6 -0.02 -0.04 -0.8 -0.06 -1 -0.08 -1.2 -0.1 -0.12 -1.4 -0.14 -1.6 -0.16 -1.8 -0.18 -0.2 -2 C -0.22 -2.2 -0.24 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 -0.05 0 0.05 0.1 0.15 0.2 PC1 PC1 Fig.
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