ELECTROPHORESIS

Supporting Information for Electrophoresis DOI 10.1002/elps.200406079

Michalis Aivaliotis, Carsten Corvey, Irene Tsirogianni, Michael Karas and Georgios Tsiotis

Membrane proteome analysis of the green-sulfur bacterium Chlorobium tepidum

ã 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 0001±0010 1

6 Addendum Table 1. List of identified proteins separated in 2-D gel with Triton X-100

No. Protein definition Accession Predicted Peptides Peptide TM GRAVYSignalP Aro- Predicted b) No./gene Mr/pI matched coverage a-heli- matic localization (%) cesa) anchor

Transport and binding proteins 1 ArsA ATPase family protein Q8KDR5/CT0980 44541/5.0 17 48 1 20.117 ± ± Inner membrane 2 Outer membrane efflux Q8KED1/CT0758 52716/8.9 8 16 3 20.163 ± ± Outer membrane protein, putative 3 ABC transporter, periplasmic Q8KDW4/CT0930 33207/8.9 9 26 3 20.388 YES 1±25 ± Inner membrane substrate-binding protein

Energy metabolism 4 ATP synthase F1, a-subunit Q8KAW8/ATPA 56869/6.3 31 50 2 20.051 ± ± Inner membrane 5 ATP synthase, b-chain Q8KAC9/ATPD 50125/5.0 37 77 1 20.130 ± ± Inner membrane 6 ATP synthase F0, B subunit Q8KGE9/ATPF 19437/7.7 8 33 1 20.225 YES 1±39 ± Inner membrane 7 Bacteriochlorophyl A protein Q46393/FMOA 40383/7.1 34 78 ± 20.288 ± ± Inner membrane 8 Chlorosome envelope O68991/GSMH 21779/4.9 7 44 2 20.125 ± ± Chlorosome protein H 9 Chlorosome envelope O68988/GSMI 25911/7.6 11 60 2 0.082 ± ± Chlorosome protein I 10 Chlorosome envelope Q8KEN5/CSMX 23970/5.7 9 40 ± 20.155 ± ± Chlorosome protein X 11 Cytochrome b6f complex Q9F722/PETC 18733/9.2 13 62 3 0.060 YES 1±24 ± Inner membrane Rieske, iron-sulfur subunit 12 Photosystem P840 RC Q8KEP5/PSCD 16607/9.7 8 45 ± 20.558 ± ± Inner membrane protein PscD 13 NADH dehydrogenase I, Q8KEC2/NDHH 20925/8.9 12 73 1 20.052 ± ± Cytoplasmic 20 kDa subunit

Protein fate 14 ATP-dependent Cip protease, Q8KG79/CIPB-2 49654/5.3 28 62 ± 20.440 YES 1±4 ± Cytoplasmic ATP-binding subunit Cipb 15 Carboxyl-terminal protease Q8KDJ6/PRC 75483/9.7 13 20 ± 20.410 ± ± Unknown 16 Propyl oligopeptidase family Q8KCV9/CT1301 77944/6.5 27 54 2 20.428 ± ± Periplasmic protein 17 Chaperonin, 60 kDa Q8KF02/GROEL 58080/5.2 37 77 ± 20.196 ± ± Cytoplasmic 18 Heat shock protein HtpG Q8KE61/HTPG 71037/5.1 42 52 2 20.243 ± ± Cytoplasmic 19 Heat shock protein Q8KB28/CT1970 16008/4.9 15 83 1 20.562 ± ± Unknown Hsp20 family 20 Chaperone protein DnaK Q8KEP3/DNAK 68387/5.2 33 52 1 20.396 ± ± Unknown 21 Polypeptide deformylase Q8KCG7/DEF 20909/5.9 10 58 ± 20.133 ± ± Cytoplasmic 22 Type-II secretion system Q8KF93/CT0434 52852/6.1 10 34 ± 20.196 ± ± Cytoplasmic protein

Protein synthesis 23 30S ribosomal protein S4 P59129 /RPSD 23319/10.2 20 74 ± 20.628 ± ± Cytoplasmic 24 Ribosomal protein L2 Q8KAH5/RPLB 30373/10.7 24 65 ± 20.613 ± ± Cytoplasmic 25 Ribosomal protein L5 Q8KAI4/RPLE 21038/10.3 15 67 ± 20.328 ± ± Cytoplasmic 26 Ribosomal protein L6 Q8KAI6/RPLF 19598/9.9 14 66 1 20.230 ± ± Cytoplasmic 27 50S ribosomal protein L9 Q8KAM4/RPLI 16489/7.9 8 58 ± 20.275 ± ± Cytoplasmic

ã 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2 M. Aivaliotis et al. Electrophoresis 2004, 25, 0001±0010

Table 1. Continued

No. Protein definition Accession Predicted Peptides Peptide TM GRAVYSignalP Aro- Predicted b) No./gene Mr/pI matched coverage a-heli- matic localization (%) cesa) anchor

28 50S ribosomal protein L10 Q8KG17/RPLJ 19282/10.1 11 73 1 20.210 ± ± Cytoplasmic 29 Ribosomal protein L13 Q8KBK4/RPLM 16903/9.9 7 49 ± 20.558 ± ± Cytoplasmic 30 Ribosome recycling factor Q8KC49/FRR 21222/9.0 13 61 ± 20.638 ± ± Cytoplasmic

Cell envelope 31 Outer surface protein, Q8KAL2/CT2144 20056/5.1 13 42 2 0.013 YES 1±19 F Outer membrane putative 32 Outer membrane protein Q8KFR8/OMPH 20430/9.5 22 67 1 20.448 YES 1±40 ± Outer membrane OmpH 33 Peptidoglycan-associated Q8KEP8/CT0638 17850/4.8 10 62 2 20.334 ± ± Outer membrane lipoprotein

Unknown function 34 Pentapeptide repeat family Q8KAT8/CT2067 46535/6.6 16 36 3 20.149 ± ± Unknown protein 35 WD-repeat family protein Q8KB12/CT1986 35900/8.0 23 63 1 20.239 YES 1±8 ± Unknown 36 Hemaglutinin-related protein Q8KGA0/CT0068 22146/4.8 15 55 ± 20.208 ± F Outer membrane

Central intermediary metabolism 37 Sulfide dehydrogenase, Q8KDM1/CT1025 45492/7.2 18 46 1 20.137 ± ± Periplasmic flavoprotein subunit, putative 38 Polysulfide reductase, Q8KAC2/CT2241 28963/8.1 12 56 1 20.345 YES 1±31 ± Cytoplasmic subunit B, putative 39 Sulfide-quinone reductase, Q8KG51/CT0117 53974/6.7 9 11 5 20.215 ± ± Inner membrane putative 40 Nitrogenase molybdenium- Q8KC89/NIFD 60680/6.1 14 32 3 20.336 ± ± Unknown iron protein, a-subunit

Biosynthesis of cofactors, prosthetic groups and carriers 41 Zeta-carotene desaturase Q8KCK1/CT1414 50979/6.7 19 35 3 20.229 ± ± Unknown

Cellular processes 42 Cell division protein FtsZ Q8KGD8/FTSZ 46167/5.9 20 41 1 20.220 ± ± Cytoplasmic/ inner membrane 43 Bacterioferritin comigratory Q8KEM5/BCP-1 17815/6.5 18 79 ± 20.551 ± ± Unknown protein, thiol peroxidase, putative 44 Bacterioferritin comigratory Q8KAZ7/BCP-2 16354/5.1 7 46 ± 0.022 ± ± Unknown protein, thiol peroxidase, putative 45 Peroxiredoxin, putative Q8KBN8/CT1747 24047/6.1 17 70 1 20.236 ± ± Cytoplasmic

Purines, pyriminides, nucleosides, and nucleotides 46 Adeninephosphoribosyl- Q8KFM9/APT 19319/5.3 5 48 2 0.100 ± ± Cytoplasmic transferase

DNA metabolism 47 Regulatory protein RecX Q8KBK8/CT1778 17890/9.5 7 42 ± 20.790 ± ± Cytoplasmic

ã 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 0001±0010 Proteomic analysis of C. tepidum 3

Table 1. Continued

No. Protein definition Accession Predicted Peptides Peptide TM GRAVYSignalP Aro- Predicted b) No./gene Mr/pI matched coverage a-heli- matic localization (%) cesa) anchor

Regulatory functions 48 Transcriptional regulator, Q8KDZ1/CT0903 27147/7.6 8 30 ± 20.253 ± ± Cytoplasmic putative

Conserved hypothetical proteins 49 Conserved hypothetical Q8KAK9/CT2147 42377/5.3 31 58 2 20.228 ± ± Unknown protein 50 Conserved hypothetical Q8KG37/CT0131 33752/5.9 21 52 1 0.043 ± ± Unknown protein 51 Conserved hypothetical Q8KBC2/CT1867 24297/8.8 16 69 1 20.247 YES 1±17 ± Unknown protein

Hypothetical proteins 52 Hypothetical protein Q8KA97/CT2270 21451/8.9 11 44 1 0.115 YES 1±25 Y Outer membrane 53 Hypothetical protein Q8KCV6/CT1304 21332/6.0 11 59 1 20.153 ± ± Unknown 54 Hypothetical protein Q8KBI6/CT1801 32338/6.3 23 63 1 20.178 YES 1±20 ± Unknown 55 Hypothetical protein Q8KDH0/CT1080 28665/6.9 10 44 2 20.024 YES 1±23 ± Unknown 56 Hypothetical protein Q8KE01/CT0893 42440/8.6 17 54 3 20.409 YES 1±44 F Outer membrane 57 Hypothetical protein Q8KBI3/CT1804 42811/5.6 28 74 2 20.248 YES 1±36 F Outer membrane 58 Hypothetical protein Q8KAL8/CT2138 19418/5.4 6 27 ± 20.455 ± ± Unknown a) Predicted by TMtrep program b) Predicted by PSORT algorithm

Table 2. List of identified proteins separated in 2-D gel with SDS and acetone treatment

No. Protein definition Accession Predicted Peptides Peptides TM SignalP GRAVYAro- Predicted b) No./gene Mr/pI matched coverage a-heli- matic localization (%) cesa) anchor

Transport and binding proteins 1 Toluene transport protein, Q8KAD3/CT2230 49260/6.0 19 49 3 YES 1±23 20.083 ± Inner membrane putative 2 ABC-type export system, Q8KEY0/CT0552 26711/5.5 21 55 1 ± 20.205 ± Inner membrane ATP-binding subunit 3 ABC-type export system, Q8KEX9/ CT0553 34024/9.9 7 25 2 YES 1±20 20.076 ± Inner membrane membrane fusion 4 Outer membrane efflux Q8KED1/CT0758 52716/8.9 12 31 3 YES 1±42 20.163 ± Ouuter membran protein, putative 5 ArsA ATPase family protein Q8KDR5/CT0980 44541/5.0 22 64 1 ± 20.117 ± Inner membrane 6 ArsA ATPase family protein Q8KAT5/CT2070 44671/5.1 11 41 ± ± 20.226 ± Inner membrane 7 Phosphate ABC transporter, Q8KDZ9/CT0895 28379/9.5 14 63 1 YES 1±24 0.020 ± Inner membrane periplasmic substrate- binding protein, putative

Energy metabolism 30 NADH dehydrogenase I, Q8KEB8/NDHI 23569/7.4 13 44 1 ± 20.388 ± Cytoplasmic 23 kDa subunit or CT0771

ã 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4 M. Aivaliotis et al. Electrophoresis 2004, 25, 0001±0010

Table 2. Continued

No. Protein definition Accession Predicted Peptides Peptides TM SignalP GRAVYAro- Predicted b) No./gene Mr/pI matched coverage a-heli- matic localization (%) cesa) anchor

31 Thioredoxin reductase Q8KE48/TRXB 34049/5.2 19 71 1 ± 20.104 ± Cytoplasmic or CT0842 32 Cytochrome b6f complex Q9F722/PETC 18864/9.2 9 50 3 YES 1±19 0.060 ± Inner membrane Rieske, iron-sulfur subunit or CT0302 33 Transketolase, C-terminal Q8KE86/CT0804 35681/5.7 18 66 1 ± 20.023 ± Cytoplasmic subunit 34 Enolase Q8KB35/ENO-1 46383/5.8 25 55 ± ± 20.306 ± Cytoplasmic or CT1962 35 2,3-Bisphosphoglycerate- Q8KFC8/GPMA or 28434/6.1 12 45 ± ± 20.552 ± Cytoplasmic dependent phosphoglycerate GPM or CT0399 mutase 36 ATP synthase, b-chain Q8KAC9/ATPD 50125/5.0 29 81 1 ± 20.130 ± Inner membrane or CT2234 37 ATP synthase F1, a-subunit Q8KAW8/ATPA 56848/6.3 36 58 2 ± 20.051 ± Inner membrane or CT2033 38 Chlorosome envelope Q8KEN5/CSMX 23970/5.7 12 55 ± ± 20.155 ± Chlorosome protein X or CT0652 39 Chlorosome envelope O68988/CSMI 25929/7.6 11 45 2 ± 0.082 ± Chlorosome protein I or CT1382 40 Chlorosome envelope O68991/CSMH 21779/5.0 12 67 2 ± 20.125 ± Chlorosome protein H or CT1417 41 Bacteriochlorophyl A protein Q46393/FMOA 40383/7.1 34 86 ± ± 20.288 ± Inner membrane or CT1499 42 Carbohydrate kinase, Q8KDR9/CT0976 34608/4.5 22 66 2 ± 20.187 ± Unknown PfkB family 43 NAD-dependent epimerase/ Q8KFU2/CT0230 39061/5.6 9 43 2 ± 20.244 ± Unknown dehydratase family protein 44 Alcohol dehydrogenase, Q8KDA2/CT1152 40657/5.4 10 33 2 ± 0.015 ± Inner membrane iron-containing 45 Succinyl-CoA synthetase, Q8KFQ3/SUCD 31076/ 5.8 13 57 1 ± 0.152 ± Cytoplasmic a-subunit or CT0269

46 Succinyl-CoA synthetase Q8KFE7/SUCC 42217/5.4 12 33 1 ± 20.082 ± Cytoplasmic b-chain or CT0380 47 Citrate lyase, subunit 1 Q8KDG1/CT1089 43776/5.2 19 68 1 ± 20.076 ± Cytoplasmic

Protein fate 8 Outer membrane protein, Q8KG61/CT0107 47365/6.6 25 54 1 ± 20.361 ± Outer membrane putative 9 Type-II secretion system Q8KF93/CT0434 52852/6.1 28 66 ± ± 20.196 ± Cytoplasmic protein 10 LipD protein, putative Q8KAV2/CT2049 50088/8.8 21 52 1 YES 1±25 20.355 ± Outer membrane 11 Trigger factor Q8KB57/TIG 48608/4.8 31 66 ± ± 20.614 ± Unknown or CT1934 12 Chaperonin, 60 kDa Q8KF02/GROEL 58080/5.2 45 66 ± ± 20.196 ± Cytoplasmic or CT0530 13 Chaperonin protein DnaK Q8KEP3/DNAK 68387/5.2 34 55 1 ± 20.396 ± Unknown or CT0643

ã 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 0001±0010 Proteomic analysis of C. tepidum 5

Table 2. Continued

No. Protein definition Accession Predicted Peptides Peptides TM SignalP GRAVYAro- Predicted b) No./gene Mr/pI matched coverage a-heli- matic localization (%) cesa) anchor

14 Peptidyl-propyl cis-trans Q8KAA2/SURA 48563/7.0 39 74 1 YES 1±22 20.290 ± Periplasmic isomerase SurA or CT2264 15 Chaperone protein htpG Q8KE61/HTPG 71037/4.8 24 45 2 ± 20.243 ± Cytoplasmic or CT0829 16 Serine protease Q8KCH4/CT1447 53528/9.3 26 49 1 YES 1±24 0.005 ± Periplasmic 17 Carboxyl-terminal protease Q8KDJ6/PRC 75483/9.7 47 62 ± ± 20.410 ± Unknown or CT1054 18 Methionine aminopeptidase Q8KAJ2/MAP 28190/6.3 13 71 ± ± 20.215 ± Unknown or CT2168 Fatty acid and phospholipid metabolism 19 Acetyl-CoA carboxylase, Q8KG13/ACCC 49181/5.9 25 61 1 ± 20.198 ± Cytoplasmic biotin carboxylase or CT0157 20 Acetyl-CoA carboxylase, Q8KG12/ACCB 17022/4.5 6 54 1 0.021 ± Cytoplasmic biotin carboxyl carrier or CT0158 protein 21 Enoyl-(acyl-carrier-protein) Q8KFH6/FABI 32445/5.9 9 28 ± ± 20.251 ± Cytoplasmic reductase or CT0350 22 Lysophospholipase L2, Q8KA96/CT2271 28997/7.1 16 77 1 ± 0.039 ± Unknown putative

Amino acid biosynthesis 23 Phosphoribosylformimino-5- Q8KF55/HISA 28711/6.0 13 57 ± ± 20.135 ± Cytoplasmic aminoimidazole carboxamide or CT0477 ribotide isomerase 24 ATP phosphoribosyl- Q8KB10/HISG 32103/5.5 7 35 ± YES 1±7 20.074 ± Cytoplasmic transferase or CT1988 25 Diaminopimelate epimerase Q8KAX9/DAPF 28052/5.3 10 59 1 ± 20.167 ± Cytoplasmic or CT2021 26 5,10-Methylenetetrahydrofolate Q8KCP5/METF 32772/5.3 8 44 1 ± 20.233 ± Cytoplasmic reductase or CT1368 27 Aspartate-semialdehyde Q8KB62/ASD 36840/4.9 28 80 ± ± 20.049 ± Cytoplasmic dehydrogenase or CT1928

28 Glutamine synthetase Q8KCK4/GLNA 78551/5.1 12 27 2 ± 20.159 ± Cytoplasmic or CT1411 29 Shikimate 5-dehydrogenase Q8KBH8/AROE 31733/5.7 20 78 2 ± 0.144 ± Cytoplasmic or CT1809

Central intermediary metabolism 48 Oxidoreductase, short-chain Q8KG22/CT0148 28662/8.8 10 57 2 ± 0.322 ± Unknown dehydrogenase/reductase family 49 Sulfur oxidation protein SoxA Q8KDM7/SOXA 32134/9.2 11 39 1 YES 1±27 20.486 ± Periplasmic or CT1019 50 Sulfur oxidation protein SoxB Q8KDM5/SOXB 68234/6.9 40 63 2 ± 20.265 ± Periplasmic or CT1021 51 Adenylylsulfate reductase, Q8KE27/APSA 73418/5.8 13 26 3 ± 20.372 ± Unknown a-subunit or CT0865

ã 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 6 M. Aivaliotis et al. Electrophoresis 2004, 25, 0001±0010

Table 2. Continued

No. Protein definition Accession Predicted Peptides Peptides TM SignalP GRAVYAro- Predicted b) No./gene Mr/pI matched coverage a-heli- matic localization (%) cesa) anchor

Biosynthesis of cofactors, prosthetic groups, and carriers 52 Porphobilinogen deaminase Q8KCJ4/HEMC 34394/6.0 26 64 1 ± 20.122 ± Cytoplasmic or CT1427 53 Magnesium-protoporphyrin IX Q8KB38/BCHM 26015/9.4 7 32 1 ± 20.189 ± Cytoplasmic monomethyl ester oxidative or CT1958 cyclase, 25 kDa subunit 54 Thioredoxin Q8KD40/CT1215 30037/4.9 13 63 ± ± 20.169 F Unknown 55 Light-independent proto- Q9F715/BCHB 58968/5.4 16 39 ± ± 20.139 ± Cytoplasmic chlorophyllide reductase or CT2151 subunit B 56 Thiamine-phosphate Q8KD79/THIE 21474/5.7 10 81 ± YES 1±4 0.081 ± Unknown pyrophosphorylase or CT1175

Cellular processes 57 General stress protein Ctc Q8KCQ1/CTC 21531/5.3 13 76 ± ± 20.111 ± Cytoplasmic or CT1362 58 Cell division protein FtsZ Q8KGD8/FTSZ 46081/5.9 42 82 1 ± 20.220 ± Cytoplasmic/ or CT0030 inner membrane 59 Cell division ATP-binding Q8KEU2/FTSE 26496/9.4 7 39 ± ± 20.062 ± Inner membrane protein FtsE or CT0590 60 Probable thiol peroxidase Q8KED5/TPX 17714/4.7 6 49 ± ± 0.26 ± Unknown or CT0754 61 Peroxiredoxin, putative Q8KBN8/CT1747 24047/6.5 22 77 1 ± 20.236 ± Unknown

Protein synthesis 62 Ribosomal protein S1 Q8KFN4/RPSA 61516/5.1 40 58 1 ± 20.171 ± Cytoplasmic or CT0288 63 30S ribosomal protein S2 Q8KBK6/RPSB 28158/5.5 12 35 ± ± 20.136 ± Cytoplasmic or CT1781 64 30S ribosomal protein S3 Q8KAH8/RPSC 28453/10.1 17 66 1 ± 20.555 ± Cytoplasmic or CT2183 65 30S ribosomal protein S4 Q8KAJ7/RPSD 23319/10.2 17 80 ± ± 20.628 ± Cytoplasmic or CT2163

66 50S ribosomal protein L1 Q8KG18/RPLA 24798/9.3 9 46 1 ± 20.160 ± Cytoplasmic or CT0152 67 Ribosomal protein L2 Q8KAH5/RPLB 30373/10.7 14 41 ± ± 20.613 ± Cytoplasmic or CT2186 68 Ribosomal protein L3 Q8KAH2/RPLC 22183/9.9 12 61 ± ± 20.387 ± Cytoplasmic or CT2189 69 Ribosomal protein L4 Q8KAH3/RPLD 22643/10.6 11 57 ± ± 20.323 ± Cytoplasmic or CT2188 70 Ribosomal protein L5 Q8KAI4/RPLE 21038/10.3 18 79 ± ± 20.328 ± Cytoplasmic or CT2177 71 Ribosomal protein L6 Q8KAI6/RPLF 19598/9.9 14 56 1 ± 20.230 ± Cytoplasmic or CT2174 72 Ribosomal protein L15 Q8KAJ0/RPLO 20083/10.1 11 61 ± ± 20.445 ± Cytoplasmic or CT2170

ã 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 0001±0010 Proteomic analysis of C. tepidum 7

Table 2. Continued

No. Protein definition Accession Predicted Peptides Peptides TM SignalP GRAVYAro- Predicted b) No./gene Mr/pI matched coverage a-heli- matic localization (%) cesa) anchor

73 Ribosomal protein L17 Q8KAJ9/RPLQ 17400/10.3 11 51 ± YES 1±17 20.752 ± Cytoplasmic or CT2161 74 Elongation factor P Q8KG11/EFP 21087/5.1 16 74 ± YES 1±10 20.304 ± Cytoplasmic or CT0159 75 Translation elongation Q8KG26/FUSA-2 76538/5.6 27 49 2 ± 20.160 ± Cytoplasmic factor G or CT0144 76 Elongation factor Ts Q8KBK7/TSF 31544/5.2 18 63 ± ± 20.305 ± Cytoplasmic or CT1780 77 Elongation factor Tu Q8KAH0/TUF 42899/5.3 16 60 1 ± 20.189 ± Cytoplasmic or CT2191 Purines, pyriminides, nucleosides, and nucleotides 78 Adenine phosphoribosyl- Q8KFM9/CT0293 19319/5.3 11 76 2 ± 0.100 ± Cytoplasmic transferase 79 Ribose-phosphate Q8KCQ2/PRSA 35265/ 6.0 17 60 ± ± 0.023 ± Cytoplasmic pyrophosphokinase or CT1361 80 Phosphoribosylformyl- Q8KCS5/PURQ 25269/5.9 10 58 ± ± 20.05 ± Cytoplasmic glycinamidine synthase I or CT1338 81 Adenylate kinase Q8KD69/ADK 23621/6.1 18 67 ± ± 20.206 ± Cytoplasmic or CT1185 82 Uridylate kinase P59003/PYRH 26138/6.2 9 46 ± ± 0.038 ± Cytoplasmic or CT1779 83 Nucleoside diphosphate Q8KAZ6/NDK 15446/5.3 9 61 ± ± 20.117 ± Cytoplasmic kinase or CT2002 DNA metabolism 84 Single-strand binding Q8KAM2/SSB-2 16992/5.5 7 57 ± ± 20.842 ± Cytoplasmic protein or CT2134 85 Single-strand binding Q8KB47/SSB-1 17804/6.7 6 46 ± ± 20.888 F Cytoplasmic protein or CT1947 86 ATP-dependent DNA Q8KC51/RECG 78649/9.2 12 19 1 ± 20.273 ± Inner membrane helicase RecG or CT1575 Cell envelope 87 Rod shape-determining Q8KEY5/MREB-1 36893/7.9 22 66 1 ± 0.067 YUnknown protein MreB or CT0547

88 OmpA family protein Q8KCR0/CT1353 24334/9.4 17 44 3 YES 1±24 20.196 ± Outer membrane 89 Outer surface protein, Q8KAL2/CT2144 20056/5.7 6 21 2 YES 1±19 0.013 F Outer membrane putative 90 Bacterial surface antigen Q8KFQ6/CT0266 93746/7.3 37 44 6 YES 1±30 20.398 ± Outer membrane family protein Regulatory functions 91 Transcriptional regulator, Q8KDZ1/CT0903 27147/7.6 9 40 ± ± 20.253 ± Cytoplasmic putative 92 Iron-dependent repressor Q8KBP8/CT1737 29952/6.0 6 31 ± ± 20.359 ± Cytoplasmic 93 Transcription antitermination Q8KG20/NUSG 21580/9.0 9 60 ± ± 20.281 ± Cytoplasmic protein NusG or CT0150 94 Transcription elongation Q8KCH5/GREA 17949/4.9 11 79 ± ± 20.481 ± Cytoplasmic factor greA or CT1446

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Table 2. Continued

No. Protein definition Accession Predicted Peptides Peptides TM SignalP GRAVYAro- Predicted b) No./gene Mr/pI matched coverage a-heli- matic localization (%) cesa) anchor

95 N-utilization substance Q8KFT2/CT0240 60221/5.4 18 45 ± ± 20.676 ± Cytoplasmic protein A, putative Unknows function 96 Hemaglutinin-related protein Q8KGA0/CT0068 22145/4.6 16 56 ± ± 20.208 F Outer membrane 97 Pentapeptide repeat family Q8KAT8/CT2067 46535/6.6 32 46 3 YES 1±25 20.149 ± Outer membrane protein 98 WD-repeat family protein Q8KB12/CT1986 35900/8.1 20 74 ± YES 1±8 20.239 ± Unknown 99 Tia invasion determinant- Q8KF15/CT0517 23521/4.6 8 47 2 YES 1±20 0.023 ± Outer membrane related protein Hypothetical proteins 100 Hypothetical protein Q8KFD2/CT0395 28475/4.9 15 64 1 YES 1±21 20.183 ± Unknown 101 Hypothetical protein Q8KF61/CT0471 30375/4.4 11 43 2 YES 1±41 20.167 F Outer membrane 102 Hypothetical protein Q8KE62/CT0828 36009/9.2 13 50 5 YES 1±55 20.123 ± Inner membrane 103 Hypothetical protein Q8KE01/CT0893 42409/8.6 14 74 3 YES 1±44 20.409 F Outer membrane 104 Hypothetical protein Q8KDR1/CT0984 24885/4.9 14 59 2 YES 1±25 20.259 F Outer membrane 105 Hypothetical protein Q8KD97/CT1157 25394/7.7 10 66 2 YES 1±20 20.328 ± Unknown 106 Hypothetical protein Q8KBP0/CT1745 25008/4.6 23 79 1 YES 1±25 20.319 F Outer membrane 107 Hypothetical protein Q8KBI6/CT1801 32338/6.3 16 58 1 YES 1±20 20.178 ± Unknown 108 Hypothetical protein Q8KBI3/CT1804 42811/5.6 29 76 2 YES 1±36 20.248 F Outer membrane Conserved hypothetical proteins 109 Conserved hypothetical protein Q8KG37/CT0131 33752/5.9 14 60 1 ± 0.043 ± Unknown 110 Conserved hypothetical protein Q8KG32/CT0136 28184/4.5 8 24 1 ± 20.033 ± Unknown 111 Conserved hypothetical protein Q8KFS6/CT0246 33714/5.3 6 25 ± ± 20.374 ± Unknown 112 Conserved hypothetical protein Q8KFQ7/CT0265 22684/4.7 9 68 ± ± 20.336 ± Unknown 113 Conserved hypothetical protein Q8KDT6/CT0958 27878/5.7 8 52 1 YES 1±15 20.587 ± Unknown 114 Conserved hypothetical protein Q8KBH3/CT1814 25395/5.4 11 50 ± ± 20.291 ± Unknown 115 Conserved hypothetical protein Q8KBC2/CT1867 24297/8.8 13 55 1 YES 1±17 20.247 ± Unknown 116 Conserved hypothetical protein Q8KDC2/CT1132 32883/6.2 14 55 ± ± 20.405 ± Unknown 117 Conserved hypothetical protein Q8KAK9/CT2147 42377/5.3 25 82 2 ± 20.228 ± Unknown a) Predicted by TMtrep program b) Predicted by PSORT algorithm

ã 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Electrophoresis 2004, 25, 0001±0010 Proteomic analysis of C. tepidum 9

Table 3. List of identified proteins separated in 2-D gel with acidic extraction with accession number, protein name, pre-

dicted Mr/pI, peptides matched and coverage (%), transmembrane (TM) a-helices predicted by Tmpred, GRAVY value, signal sequence predicted by SignalP, presence of aromatic anchor in the C-terminal, and predicted cel- lular localization by PSORT

No. Protein definition Accession Predicted Peptides Peptide TM GRAVYSignalP Aromatic Predicted b) No./gene Mr/pI matched coverage a-heli- anchor localization (%) ces

Transport and binding proteins 1 ABC transporter, periplasmic Q8KEL8/CT0670 31397/9.0 27 83 1 20.077 YES 1±25 ± Inner membrane substrate-binding protein 2 Phosphate ABC transporter, Q8KDZ9/CT0895 28379/9.5 20 80 1 0.020 YES 1±24 ± Inner membrane periplasmic substrate- binding protein 3 ArsA ATPase family protein Q8KB52/CT1939 42960/6.1 13 38 ± 20.043 ± ± Inner membrane 4 TolB protein (precursor) Q8KEQ0/TOLB 47275/9.2 13 34 2 20.285 YES 1±27 ± Periplasmic

Energy metabolism 5 Bacteriochlorophyl A protein Q46393/FMOA 40383/7.1 28 67 ± 20.288 ± ± Inner membrane 6 NADH dehydrogenase I, Q8KEB8/NDHI 23569/7.4 8 47 1 20.388 ± ± Cytoplasmic 23 kDa subunit 7 Aconitate hydratase Q8KEY9/ACN 92163/5.0 11 16 5 20.045 ± ± Cytoplasmic 8 Isocitrate dehydrogenase, Q8KFH5/ICD 80838/5.2 12 18 2 20.186 ± ± Cytoplasmic NADP-dependent, monomeric type 9 Phosphoenolpyruvate Q8KAD1/PCKG 69932/5.4 16 23 3 20.333 ± ± Cytoplasmic carboxykinase (GTP)

Protein fate 10 Chaperonin, 60 kDa Q8KF02/GROEL 58080/5.2 21 45 ± 20.196 ± ± Cytoplasmic 11 Peptidyl-prolyl cis-trans Q8KAA2/SURA 48563/7.0 9 23 1 20.290 YES 1±22 ± Periplasmic isomerase SurA 12 ATP-dependent Clp protease, Q8KFC3/CLPX 48331/6.6 11 28 1 20.215 ± ± Cytoplasmic ATP-binding subunit Clpx 13 Preprotein translocase SecA Q8KD18/SECA 118004/5.8 15 24 1 20.553 ± ± Cytoplasmic, subunit inner membrane

Protein synthesis 14 Elongation factor P Q8KG11/EFP 21087/5.1 16 62 ± 20.304 YES 1±10 ± Cytoplasmic 15 Peptide chain release factor 3 Q8KE72/PRFC 60720/5.5 11 21 20.408 ± ± Cytoplasmic 16 Ribosomal protein S1 Q8KFN4/RPSA 61516/5.1 13 26 1 20.171 ± ± Cytoplasmic Cell envelope

17 N-Acetylmuramoyl-L-alanine Q8KGB4/AMIB 60430/8.5 15 39 3 20.383 ± ± Periplasmic amidase 18 OmpA family protein Q8KCR0/CT1353 24334/9.4 11 52 3 20.196 YES 1±24 ± Outer membrane

Unknown function 19 Pentapeptide repeat family Q8KAT8/CT2067 46535/6.6 24 42 3 20.149 YES 1±25 ± Periplasmic protein

Central intermediary metabolism 20 Sulfide dehydrogenase, Q8KDN1/FCCB-1 46240/8.6 24 43 5 20.082 YES 1±32 ± Periplasmic flavoprotein subunit 21 Sulfur oxidation protein Sox A Q8KDM7/SOXA 32398/9.2 16 60 1 20.486 YES 1±27 ± Periplasmic

ã 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 10 M. Aivaliotis et al. Electrophoresis 2004, 25, 0001±0010

Table 3. Continued

No. Protein definition Accession Predicted Peptides Peptide TM GRAVYSignalP Aromatic Predicted b) No./gene Mr/pI matched coverage a-heli- anchor localization (%) ces

22 Hydrolase, haloacid Q8KGF4/CT0014 25419/4.9 6 27 1 0.080 ± ± Cytoplasmic dehalogenase-like family Transcription 23 N-Utilization substance Q8KBS4/NUSB 19214/8.8 9 48 ± 20.541 ± ± Cytoplasmic protein B homolog Biosynthesis of cofactors, prosthetic groups and carriers 24 Uroporphyrinogen-III synthase Q8KCJ3/HEMD 26616/4.8 7 32 1 0.096 ± ± Uknown 25 CrtF-related protein Q8KGE0/CT0028 37934/5.1 10 47 1 20.140 ± ± Cytoplasmic 26 Thiamine biosynthesis Q8KCH9/THIC 61331/6.2 14 30 1 20.303 ± ± Cytoplasmic protein thiC Purines, pyriminides, nucleosides, and nucleotides 27 Dihydroorotate dehydrogenase, Q8KB48/PYRDII 27562/6.1 7 32 4 0.248 ± ± Inner membrane electron transfer subunit Amino acid biosynthesis 28 Cysteine synthase/cystathionine Q8KB68/CT1921 42944/5.1 8 26 4 20.104 ± ± Uknown b-synthase family protein 29 Acetolactate synthase, Q8KER5/ILVB 62874/5.8 11 26 5 20.081 ± ± Uknown large subunit DNA metabolism 30 Primosomal protein N' Q8KD67/PRIA 91101/8.9 17 31 2 20.192 ± ± Cytoplasmic Conserved hypothetical proteins 31 Conserved hypothetical protein Q8KG32/CT0136 28184/4.8 11 59 1 20.033 ± ± Uknown 32 Conserved hypothetical protein Q8KET2/CT0600 20803/7.8 14 76 1 20.104 YES 1±26 ± Periplasmic 33 Conserved hypothetical protein Q8KD50/CT1205 26680/5.4 12 46 1 0.110 YES 1±18 ± Uknown 34 Conserved hypothetical protein Q8KBZ3/ CT1637 27024/4.8 8 43 2 0.027 ± ± Cytoplasmic Hypothetical proteins 35 Hypothetical protein Q8KE01/CT0893 42440/8.6 10 30 3 20.409 YES 1±44 F Outer membrane 36 Hypothetical protein Q8KBI3/CT1804 42811/5.6 18 62 2 20.248 YES 1±36 F Outer membrane 37 Hypothetical protein Q8KB89/CT1900 43375/7.2 12 43 2 20.416 YES 1±23 ± Uknown a) Predicted by TMtrep program b) Predicted by PSORT algorithm

ã 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim