Proteome of Methanosarcina acetivorans Part I: An Expanded View of

Proteome of Methanosarcina acetivorans Part I: An Expanded View of the Biology of the Cell Qingbo Li,† Lingyun Li,‡ Tomas Rejtar,‡ Barry L. Karger,*,‡ and James G. Ferry*,† Center for Microbial Structural Biology, Department of Biochemistry and Molecular Biology, 205 South Frear Laboratory, Penn State University, University Park, Pennsylvania 16802 and Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115 Received September 21, 2004

Methanosarcina acetivorans is representative of the genus that is distinguished from all other methaneproducing genera by extensive metabolic diversity predicted from the large genome. In Part I of this study, two-dimensional gel electrophoresis and MALDI-TOF-TOF mass spectrometry was used to investigate the proteome of methanol- or acetate-grown M. acetivorans, with the goal of an initial characterization of the diversity of the proteins synthesized. A total of 412 proteins were identified, representing nearly 10% of the ORFs, with nearly 30% conserved hypothetical or hypothetical. Of the 412 proteins, 188 were found in both acetate- and methanol-grown cells, 122 were detected only in acetate-grown cells, and 102 only in methanol-grown cells. The results revealed the expression of a remarkable number of redundant genes which encode enzymes involved in the pathways for methanogenesis from methanol or acetate, suggesting an important role for the unusually high percentage of redundant genes in Methanosarcina species. Evidence was obtained for synthesis of a sodium-transporting oxidoreductase in acetate-grown cells, with the potential to function in energy conservation. Several transcriptional regulatory proteins were identified that also function in the Bacteria domain, raising questions regarding their interaction with the Archaea/Eucarya-type basal transcription apparatus. In addition, a significant number of proteins involved in protein folding were shown to be synthesized in methanol- and acetate-grown cells. These studies provide the first examination of the protein diversity of M. acetivorans. Keywords: proteomics • 2-D electrophoresis • MALDI-TOF-TOF MS • Methanosarcina acetivorans • methanol • acetate • methanogenesis • Archaea

Introduction All of life is classified into three domains (Archaea, Bacteria, and Eucarya) of which the Archaea is the least understood. Methane-producing microbes, the largest known group representing the Archaea, are essential to the global carbon cycle.1 Methane and carbon dioxide are the final products of the microbial decomposition of organic matter in a diversity of oxygen-free (anaerobic) habitats, such as the rumen of cattle, sewage treatment plants, rice paddies, and natural wetlands. An estimated one billion metric tons of methane are produced annually.1 Most of this methane is produced by a two-step process in which complex organic matter is decomposed to acetate by fermentative microbes that is further converted to methane and carbon dioxide by methane-producing species from the genera Methanosarcina and Methanosaeta. This two-step process also applies to the controlled decomposition of renewable biomass to methane (biomethanation), * To whom correspondence should be addressed. B.L.K.: Tel: (617) 3732867. Fax: (617) 373-2867. E-mail: [email protected]. J.G.F.: Tel: (814) 8635721. Fax: (814) 863-6217. E-mail: [email protected]. † Center for Microbial Structural Biology, Department of Biochemistry and Molecular Biology, 205 South Frear Laboratory, Penn State University. ‡ Barnett Institute and Department of Chemistry, Northeastern University.

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a potentially important nonpolluting energy source in the face of dwindling supplies of fossil fuels. Significantly, the conversion of acetate to methane is the rate-limiting step, of which relatively little is understood. A comprehensive understanding of the physiology, biochemistry and molecular biology of acetate conversion to methane is thus necessary to identify the genetic and biochemical parameters that, when manipulated, will enhance the rate and control the reliability of biomethanation. This level of understanding of acetate conversion to methane depends on the identification of proteins essential for the process which begins with characterization of the proteome. Methanosarcina acetivorans was isolated from a near-shore sub-marine canyon where it participates in the decomposition of giant kelp (Macrocystis pyrifera) to methane.2 The organism has a specific function, generating methane from the acetate that is produced by other microbes which decompose the kelp. M. acetivorans is ideally suited for characterization of the proteome of an acetate-utilizing methanogenic species as it has a tractable genetic exchange system,3 and the genome sequence is available.4 Furthermore, the genome of M. acetivorans at 5 751 492 base pairs is the largest yet reported for any of the Archaea, reflecting the expanse of metabolic diversity yet to 10.1021/pr049832c CCC: $30.25

 2005 American Chemical Society

Proteome of Methanosarcina acetivorans Part I

be discovered. The M. acetivorans genome is annotated with a substantial number of duplicated genes, a feature that further distinguishes the M. acetivorans genome from all other sequenced genomes of methane-producing species.4 This extensive duplication raises questions regarding expression of the genes, and the physiological significance of the apparently redundant proteins. Herein, these redundant proteins are referred to as paralogs. Remarkably, the identification of only two proteins from M. acetivorans have up to now been reported.5,6 Indeed, a global analysis of the proteomes of only two methane-producing species has been reported,7-11 neither of which are from the genus Methanosarcina. Here, we present an initial study of the proteome of M. acetivorans grown with either acetate or methanol. Specifically, 2-dimensional gel electrophoretic (2-DE) separation of the proteins from cell lysates of acetate- and methanol-grown cells were analyzed by MALDI-TOF MS and MALDI-TOF-TOF MS, following in-gel tryptic digestion. MS/MS was particularly useful for identification of proteins that were not well separated by 2-DE. In total, 412 proteins were identified, several of which are paralogs. Although not previously assigned specific roles, several of the identified proteins have annotations which suggest potential functions in methanogenesis, the regulation of gene expression, electron transport, protein folding, stress and conservation of energy. In Part II,12 we build on the results presented here by identifying proteins that are present in different amounts between acetate- and methanol-grown M. acetivorans to further extend the biological understanding of M. acetivorans, in particular, and to the genus Methanosarcina in general.

Materials and Methods Growth. M. acetivorans was cultured anaerobically in a 14-L Microferm fermentor (New Brunswick Scientific, Edison, NJ) at 37 °C in high-salt medium, as previously described,13 with acetate (100 mM constant concentration) or methanol (250 mM initial concentration) as substrates for growth. Inocula were cultured with the respective substrates. Doubling times for acetate- and methanol-grown cultures were 84 and 42 h, respectively. Cells were harvested at mid-exponential growth corresponding to an OD600 of 0.8 and 0.6 for acetate and methanol cultures, respectively. Cells were harvested by centrifugation at 1600 × g at 4 °C and stored at -80 °C. Protein Extraction. Thawed cell pellets were resuspended in buffer containing (final concentrations) 30 mM Tris-HCl (pH 8.8), 50 mM MgCl2, 10 U/mL DNase (Roche, Indianapolis, IN), 25 mg/mL RNase (Roche) and 1 mL/mL protease inhibitor set (Roche) catalog No. 1 206 893. This procedure resulted in partial lysis. Cells were further disrupted by sonication with a Model 75T sonicator (VWR International, Bristol, CT) for 5 cycles of 30 s each at maximum power. Cell debris and unbroken cells were removed by centrifugation at 13 000 × g for 30 min at 4 °C. The supernatant was collected and mixed with (in final concentrations): urea (8 M), CHAPS (4%, w/v), DTT (60 mM), Pharmalyte (2%, v/v) (Amersham Pharmacia, Piscataway, NJ) and 1 mL/mL protease inhibitor. Protein concentrations ranged from 1 to 4 mg/mL, determined by the Bradford assay14 (BioRad, Hercules, CA) using BSA as standard. The crude protein extract was stored at -80 °C. Two-Dimensional Gel Electrophoresis (2-DE). IEF was performed with 18-cm IPG strips (pH 4-7, 6-9, or 3-10, Amersham Pharmacia). Each IPG strip was rehydrated for 16 h in 340 mL of a solution containing 8 M urea, 2% (w/v) CHAPS,

research articles 60 mM DTT, 2% (v/v) Pharmalyte and 1 mL/mL of protease inhibitor cocktail. For the pH 4-7 and 3-10 strips, 340 µg of protein was included in the rehydration solution. For IPG strips of pH 6-9, 540 µg of protein was loaded using the paper bridge method instead of at the rehydration step.15 IEF was performed with the Multiphor II unit (Amersham Pharmacia) applying the following voltage settings: 1 min gradient 0-500 V, 2 mA, 5 W; 1.5 h gradient 500-3500 V, 2 mA, 5 W; and a final phase of constant 3500 V, 2 mA, 5 W, for 7.5 h (pH 4-7 and 3-10) or 15 h (pH 6-9). The IPG strips were then equilibrated in solution A, followed by solution B (15 min each) for the reduction and alkylation of proteins. Solution A contained 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 20 mM DTT. Solution B contained 2.5% (w/v) iodoacetamide in place of DTT. The second dimension separation was performed with a precast ExcelGel SDS homogeneous (12% polyacrylamide) gel (Amersham), unless otherwise specified, using the Multiphor II unit. The electrophoresis was run at 600 V, 25 mA, and 30 W for 35 min, followed by 600 V, 18 mA and 30W for 175 min. Gels were stained with Coomassie Blue R250 (acidic), as described in the Multiphor II user manual. Gel images were generated using a Microtek ScanMaker 5900 scanner (Microtek International, Inc., Redondo Beach, CA). Analysis of images was performed using PDQuest image analysis software (BioRad).16 Protein Identifications. Protein spots were excised using a 2-mm diameter gel puncher (Amersham Pharmacia) and a scalpel. In-gel trypsin digestion and peptide extraction were performed, as previously described,17 except without the reduction and alkylation steps. The extracted peptide solutions were concentrated and desalted using C18 Ziptip (Millipore, Bedford, MA)18 prior to MS and MS/MS analysis of peptides which was performed using an AB 4700 TOF/TOF Proteomics Analyzer (Applied Biosystems, Framingham, MA). Approximately 0.6 mL of the peptide mixture from each gel spot was directly deposited onto a 192-well MALDI target plate and allowed to dry before 0.3 mL of the MALDI matrix solution was added. The solution, prepared daily, contained 7 mg/mL R-cyano-4hydroxycinnamic acid in 0.1% trifluroacetic acid and 50% (v/ v) acetonitrile. The MS spectra were obtained by accumulating 1000 laser shots, and the acquired spectra were externally calibrated using 6 calibrant positions on the MALDI plate, resulting in a mass accuracy of approximately (50 ppm. The 10 highest peaks in each MS spectrum were selected for MS/ MS analysis, and the spectra were acquired by the accumulation of 1000 laser shots. The order of MS/MS acquisition for a particular spot was from the most intense precursor ion down to the lowest. Database searches were performed using a Mascot server version 1.9 (Matrix Science, London, UK) against the database of M. acetivorans downloaded from the NCBI website [http:// www.ncbi.nlm.nih.gov/]. Combined data from the MALDI MS and MS/MS acquisitions for each spot on the MALDI sample plate were submitted to database searching using GPS Explorer software (Applied Biosystems). The mass tolerance of the precursor ion was set to (50 ppm and the MS/MS fragment ion tolerance to (0.2 Da. Up to 2 missed cleavages were allowed. The only fixed modification used in the search was carbamidomethylation of cysteine. Variable modifications included methionine oxidation, and pyro-glu (N-terminal Glu and Gln). Protein hits with a Mascot score greater than 80 (p < 0.001) were accepted automatically as identified proteins, excluding paralogs, for which no unique peptide was identified. Journal of Proteome Research • Vol. 4, No. 1, 2005 113

research articles The remaining hits with a Mascot score >49 or a statistically significant (p < 0.05) MS/MS score were subjected to manual validation. In the validation process, the peptides identified in a particular spot with a statistically significant MS/MS score were used as internal standards to recalibrate the MS spectra whenever available. Using this approach, a mass accuracy of (10 ppm was achieved for > 90% of the spectra, resulting in minimization of false positive protein identifications. The manual validation procedure was found especially useful when more than one potential protein candidate was reported by Mascot on the same MALDI spot. Gene Arrangement and Clustering. References to the arrangement and clustering of genes were obtained from the web site19 of The Institute for Genomic Research (TIGR). In some cases, references to the arrangement and clustering of genes were as reported in the published literature.

Results and Discussion Two-Dimensional Gel Analysis. In addition to acetate, M. acetivorans is able to convert several single-carbon substrates to methane, conserving energy for growth. In Part II,12 we show that M. acetivorans regulates protein synthesis in response to the growth substrate, a property that was exploited to maximize the number of proteins identified in the proteome by analysis of cells grown with either acetate or methanol. Proteins from acetate- or methanol-grown cells were separated by 2-DE according to pI and MW. When pH 3-10 IPG strips were used for the first dimension, only 12-14 spots were resolved in the pH 7-10 range for either growth condition (data not shown), although the products of 30% of the ORFs are predicted in the basic range, based on the virtual 2D gel of M. acetivorans.19 Since greater than 90% of the protein spots were detected in the pH 4-7 range, pH 4-7 IPG strips were thus mainly used for subsequent 2-DE separations. The IPG strips were loaded with 340 mg of protein from acetate- or methanol-grown cells, and the gels stained with Coomassie blue. A total of 484 or 456 spots visible to the eye were excised from the gels containing proteins from acetate- or methanol-grown cells, respectively. An additional 14 spots were excised from the pH 7-9 region of each gel utilizing pH 6-9 IPG strips that were loaded with 540 mg of protein from acetate- or methanol-grown cells. In total, 968 spots were excised for analysis. Protein Identification. The excised spots were subjected to in-gel digestion and protein identification by MALDI-MS and MS/MS analysis, and 92% of the spots generated significant protein hits representing the products of 412 genes listed in Table 1. A relatively large amount of protein was loaded onto the gels to maximize the number of detectable spots that also presented a challenge for identification of unresolved proteins. Approximately one-half of the spots had more than one protein present, and 2-3% of the spots had as many as 4 proteins present. Figure 1 shows an example of the identification of multiple proteins in a single gel spot containing rubrerythrin (MA0639), TATA-binding protein (MA4331) and archaeal transcription factor E (MA3871) with similar molecular weights (∼20kDa) and pI’s (∼5). The combination of the MS and MS/ MS spectra resulted in highly significant Mascot scores for all three proteins (Table 1), whereas using peptide mass fingerprinting (PMF) alone allowed identification of only one of the proteins (rubrerythrin). In another example, MS/MS was able to identify the products of redundant genes with high sequence similarity, as shown in Figure 2 for the identification of paralogs of the R subunit (CdhA) of CO dehydrogenase/acetyl-CoA 114

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synthase that were contained in one spot. The MS/MS data identified the sequence of unique peptides encoded by MA3860 (DVYLIAEEFLK) and MA1016 (DVYYIAEEFLK). Of the 412 identified proteins, 188 were found in both acetate- and methanol-grown cells, 122 only in acetate-grown cells, and 102 only in methanol-grown cells. Thus, a total of 310 and 290 proteins were identified in acetate- and methanolgrown cells, respectively. It should be cautioned that proteins found only in acetate- or methanol-grown cells are not necessarily unique to those cells.. In Part II, we describe the further analyses of 2-D gels that identify proteins synthesized in different amounts in response to the growth substrate. Table 2 and Figure 3 summarize the functional categorization of the 412 identified proteins based on Clusters of Orthogonal Groups (COG’s) available from the NCBI website. The proteins fall into 4 major general function categories that can be further divided into 20 sub-function categories. Of the 412 proteins, 31% belong to the “poorly characterized” category, annotated as predicted proteins, hypothetical proteins, or conserved hypothetical proteins. Pathways for Methanogenesis. M. acetivorans is capable of growth and methanogenesis from acetate and single-carbon substrates such as methanol; however, unlike M. barkeri and M. mazei, M. acetivorans is unable to reduce CO2 to methane with H2 as the electron donor.2 No enzymes have as yet been characterized from M. acetivorans that function in any pathway for methanogenesis. Figure 4 shows the principal steps in the pathways for conversion of methanol or acetate to methane, as described for other Methanosarcina species, M. barkeri, M. mazei, and M. thermophila.20-22 Figure 4 also shows the loci of genes for which products were found in M. acetivorans (Table 1), a result suggesting that M. acetivorans utilizes the same general steps in methanogenesis from acetate and methanol. Thus, the results presented here for enzymes catalyzing the steps shown in Figure 4 are likely to apply to most if not all Methanosarcina species. (i) Methanogenesis from Acetate. The M. acetivorans genome4,19 contains two gene clusters (MA1011-1016 and MA3860-3865),4,19 each predicted to encode the five subunits of CO dehydrogenase/acetyl-CoA synthase, for which the products of MA1011, MA1013-1016, and MA3860-3864 were detected in acetate-grown cells (Table 1). Both gene clusters are arranged identical to that in M. thermophila, reported to comprise an operon.23 The arrangement of genes in each cluster are consistent with coexpression in operons and synthesis of all five subunits for both paralogs of the synthase. In acetategrown M. thermophila,24 the synthase catalyzes the cleavage of acetyl-CoA (step 2, Figure 4) with transfer of the methyl group to the cofactor H4MPT, forming methyl-H4MPT. The enzyme also oxidizes the carbonyl group of acetyl-CoA to CO2, with transfer of the electrons to ferredoxin.25,26 The apparent expression of both operons in M. acetivorans is unexplained when considering they are 98% identical over a 6.6-kbp stretch consistent with no appreciable difference in enzyme function. However, duplicate operons are conserved in M. mazei,19 supporting an important role for the apparent redundancy. The synthase of acetate-grown M. thermophila comprises greater than 10% of the total cell protein;27 thus, redundancy in Methanosarcina species may be necessary to attain this level of the synthase during growth on acetate. Indeed, the conversion of acetate to methane yields very little energy to the cell for growth,28 consistent with high levels of the synthase that are necessary to achieve a high rate of conversion of acetate

research articles

Proteome of Methanosarcina acetivorans Part I

Table 1. ORFs encoding the 412 Proteins Identified in Acetate- and Methanol-Grown Cells of Methanosarcina acetivoransa,b,c Mascot scorec loci

gene annotationa

COG codeb

MA0010

formylmethanofuran-tetrahydromethanopterin N-formyltransferase pyruvate synthase, subunit β pyruvate synthase, subunit R pyruvate synthase, subunit δ pyruvate synthase, subunit γ conserved hypothetical protein peptide chain release factor arginyl-tRNA synthetase conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein myo-inositol-1-phosphate synthase Hsp60 phenylalanyl-tRNA synthetase, subunit R conserved hypothetical protein gluconate 5-dehydrogenase proliferating cell nuclear antigen conserved hypothetical protein acetylornithine aminotransferase predicted protein phosphonopyruvate decarboxylase small heat shock protein predicted protein monomethylamine methyltransferase monomethylamine corrinoid protein conserved hypothetical protein translation initiation factor 5 molybdenum cofactor biosynthesis protein B alanyl-tRNA synthetase 3-isopropylmalate dehydrogenase 3-isopropylmalate dehydratase methionyl aminopeptidase predicted protein methionine adenosyltransferase N-(5-phospho-D-ribosylformimino)-5-amino-1(5-phosphoribosyl)-4-imidazole carboxamide isomerase phosphomannomutase 4-hydroxybenzoate decarboxylase aconitate hydratase tetrahydromethanopterin methyltransferase, subunit H conserved hypothetical protein formylmethanofuran dehydrogenase, subunit F formylmethanofuran dehydrogenase, subunit A formylmethanofuran dehydrogenase, subunit C formylmethanofuran dehydrogenase, subunit D translation initiation factor 2B, subunit 2 formylmethanofuran dehydrogenase, subunit E aspartate-semialdehyde dehydrogenase fructose-bisphosphate aldolase methanol-5-hydroxybenzimidazolylcobamide co-methyltransferase, isozyme 1 methanol-5-hydroxybenzimidazolylcobamide co-methyltransferase, isozyme 1 conserved hypothetical protein cobalamin biosynthesis protein DNA mismatch repair protein predicted protein chorismate synthase glutamate-1-semialdehyde 2,1-aminomutase phosphoglycerate dehydrogenase ribosomal protein S2p metallo-β-lactamase bifunctional short chain isoprenyl diphosphate synthase pyruvate phosphate dikinase antigen groES protein (Cpn10) groEL protein (Cpn60) aspartate aminotransferase rubrerythrin

C C C C C S J J R R R R I O J S Q L S E

MA0031 MA0032 MA0033 MA0034 MA0039 MA0042 MA0043 MA0056 MA0057 MA0058 MA0059 MA0075 MA0086 MA0090 MA0092 MA0107 MA0110 MA0114 MA0119 MA0124 MA0132 MA0133 MA0134 MA0144 MA0145 MA0165 MA0182 MA0187 MA0194 MA0201 MA0202 MA0212 MA0215 MA0216 MA0218 MA0241 MA0246 MA0250 MA0269 MA0289 MA0305 MA0306 MA0307 MA0308 MA0379 MA0381 MA0430 MA0439 MA0455 MA0456 MA0516 MA0521 MA0522 MA0539 MA0550 MA0581 MA0592 MA0600 MA0605 MA0606 MA0608 MA0627 MA0630 MA0631 MA0636 MA0639

pI

AC

ME

31,783

4.83

ND

267

32,312 44,505 10,093 19,594 31,571 46,431 63,411 7,954 7,948 7,984 7,925 40,894 59,032 61,199 10,902 27,815 26,659 31,033 44,240 45,999 42,987 17,683 15,637 50,847 23,247 47,289 22,625 19,649 104,272 41,292 17,985 32,320 12,861 44,485 25,826

8.11 4.88 4.82 6.62 6.92 5.76 5.20 4.69 4.49 4.77 4.63 5.96 4.90 5.33 4.47 5.99 4.55 6.51 5.49 7.09 4.82 4.78 5.87 4.95 4.62 5.19 8.58 5.32 5.46 4.96 4.80 5.27 4.90 5.18 4.70

ND 99 93 ND 93 ND 70 159 136 288 ND 271 185 70 47 430 375 31 193 58 ND 411 240 ND 216 121 ND ND 98 108 688 67 103 196 85

65 62 153 71 ND 181 88 163 97 147 203 211 444 89 86 34 ND ND 166 ND 53 380 ND 74 ND ND 66 73 ND 149 518 111 ND 506 ND

G H C H R C C C C J C E G

49,084 46,561 103,833 34,204 25,202 38,989 65,399 30,205 14,134 33,406 23,761 36,930 28,285 51,162

5.50 5.66 5.29 4.74 4.70 5.30 5.68 5.20 4.57 5.51 5.62 5.10 5.81 4.82

89 172 142 103 42 68 390 ND ND 224 210 121 199 ND

315 151 ND 434 ND 95 64 399 216 ND 169 249 347 180

R

27,917

4.44

ND

588

K H L

18,305 36,301 73,162 15,182 39,348 46,346 55,736 27,247 49,803 36,033 97,618 75,740 12,525 58,205 43,792 18,272

4.71 6.20 6.37 5.82 6.36 5.09 5.16 4.83 5.37 4.79 5.25 5.06 5.15 5.07 5.11 5.09

51 ND ND 333 ND 61 346 ND 82 99 548 56 136 266 38 152

ND 205 123 87 113 ND 500 107 ND 64 306 ND 465 560 38 62

G O R S J H J E E J E E

E H E J R H G O O E C

MW

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Table 1. (continued) Mascot scorec loci

gene annotationa

COG codeb

MA0644 MA0652 MA0655 MA0659

translation initiation factor 2, subunit R cell division protein pelota conserved hypothetical protein Na+-transporting NADH:ubiquinone oxidoreductase, subunit 1 conserved hypothetical protein predicted protein conserved hypothetical protein sulfite reductase hypothetical protein (multidomain) predicted protein predicted protein transcriptional regulator diaminopimelate decarboxylase transcriptional regulator, ArsR family homocysteine desulfhydrase predicted protein tyrosyl-tRNA synthetase malate dehydrogenase conserved hypothetical protein predicted protein formylmethanofuran dehydrogenase, subunit C conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein HesB family protein orotate phosphoribosyltransferase conserved hypothetical protein nucleoside triphosphate (NTP):5-deoxyadenosylcobinamide phosphate nucleotidyltransferase methionine adenosyltransferase carbon monoxide dehydrogenase, subunit γ carbon monoxide dehydrogenase accessory protein carbon monoxide dehydrogenase, β subunit carbon monoxide dehydrogenase, subunit  carbon monoxide dehydrogenase, subunit R glyceraldehyde 3-phosphate dehydrogenase (phosphorylating) indolepyruvate ferredoxin oxidoreductase, subunit β NAD+ synthase (glutamine-hydrolyzing) ribosomal protein L3p ribosomal protein S3p ribosomal protein L29p ribosomal protein L5p ribosomal protein S5 ribosomal protein L30p ribosomal protein L15p adenylate kinase fructose-bisphosphatase purine NTPase conserved hypothetical protein predicted protein conserved hypothetical protein translation elongation factor 1, subunit R translation elongation factor 2 ribosomal protein L30e DNA-directed RNA polymerase, subunit A DNA-directed RNA polymerase, subunit B DNA-directed RNA polymerase, subunit H adenosylhomocysteinase chlorohydrolase family protein conserved hypothetical protein asparaginase O-linked GlcNAc transferase O-linked N-acetylglucosamine transferase predicted protein thioredoxin aspartate aminotransferase GTP-binding protein 3-isopropylmalate dehydratase predicted protein transcriptional regulator, Hth-3 family

J R S C S

MA0678 MA0679 MA0684 MA0685 MA0693 MA0702 MA0710 MA0723 MA0726 MA0750 MA0808 MA0814 MA0815 MA0819 MA0821 MA0827 MA0832 MA0841 MA0866 MA0902 MA0903 MA0919 MA0936 MA0938 MA0962 MA1011 MA1013 MA1014 MA1015 MA1016 MA1018 MA1023 MA1030 MA1072 MA1078 MA1079 MA1085 MA1092 MA1093 MA1094 MA1096 MA1152 MA1164 MA1165 MA1166 MA1171 MA1256 MA1257 MA1261 MA1262 MA1264 MA1266 MA1275 MA1276 MA1278 MA1317 MA1362 MA1364 MA1367 MA1369 MA1385 MA1390 MA1393 MA1412 MA1424 116

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C C K K E K E J C G C Q P K S F O H E C D C C C G C H J J J J J J J F G L P J J J K K K H F S E R R O E R E K

MW

pI

AC

ME

30,510 39,501 32,353 49,300

7.68 5.91 5.60 6.30

ND ND ND 204

129 59 56 ND

37,011 28,387 14,296 24,761 42,270 9,412 16,760 13,337 47,427 32,836 42,444 45,426 36,263 32,734 28,048 21,817 26,944 35,131 37,778 12,156 11,367 22,054 35,011 22,520

6.75 6.01 4.97 8.88 5.38 9.70 4.72 5.55 5.68 7.53 5.34 4.90 5.61 4.80 5.03 4.75 4.91 4.90 5.66 4.74 4.28 5.77 5.42 5.48

68 236 180 119 ND 55 ND 210 88 ND 142 43 578 ND 50 ND ND ND 390 34 225 94 ND ND

ND 58 ND ND 85 ND 96 ND 60 108 505 ND 152 66 33 146 172 277 ND ND ND 354 247 49

45,723 51,104 26,288 52,785 18,437 90,290 36,753 21,253 38,875 36,870 34,823 7,607 18,531 22,861 17,640 15,263 23,819 39,025 122,315 32,319 29,047 55,565 46,414 80,674 10,412 44,300 67,336 8,806 45,384 48,734 27,765 46,477 45,305 33,839 8,971 9,354 39,912 38,420 45,940 15,011 21,230

5.03 5.51 4.81 4.60 6.73 5.03 6.03 6.16 5.44 9.99 5.64 5.38 8.84 9.11 9.52 9.03 4.91 5.15 5.44 6.42 6.42 5.54 7.24 5.54 7.71 4.82 5.67 6.11 5.58 5.23 5.40 5.75 4.25 6.14 5.61 5.62 5.05 5.15 4.97 5.76 5.15

ND 157 92 53 82 91 162 446 527 ND 200 266 ND 64 ND ND 825 303 81 ND 119 97 148 341 188 644 190 ND 191 224 ND 123 627 316 36 60 99 ND 107 ND 228

53 85 ND 107 ND 50 243 105 ND 87 369 273 65 ND 122 146 475 309 53 37 ND ND 493 336 188 572 190 98 337 370 50 70 ND ND 120 ND 210 104 ND 326 67

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Proteome of Methanosarcina acetivorans Part I Table 1. (continued)

Mascot scorec loci

gene annotationa

COG codeb

MA1432 MA1444 MA1456 MA1460 MA1462 MA1469 MA1471 MA1477 MA1478 MA1484 MA1521 MA1524 MA1525 MA1554 MA1560 MA1567 MA1574 MA1610 MA1616

hypothetical protein (multidomain) conserved hypothetical protein predicted protein phage shock protein A universal stress protein response regulator receiver translation elongation factor 1, subunit β heat shock protein heat shock protein 70 geranylgeranyl reductase ribosomal protein L7ae nucleoside-diphosphate kinase translation initiation factor If2 conserved hypothetical protein predicted protein pyridoxine biosynthesis protein superoxide dismutase threonine synthase methanol-5-hydroxybenzimidazolylcobamide co-methyltransferase, isozyme 3 methanol-5-hydroxybenzimidazolylcobamide co-methyltransferase, isozyme 3 peptidylprolyl isomerase phosphopyruvate hydratase Hsp60 methenyltetrahydromethanopterin cyclohydrolase aspartate aminotransferase conserved hypothetical protein nitroreductase conserved hypothetical protein multicatalytic endopeptidase complex, subunit R dihydroxy-acid dehydratase methyltransferase aspartate aminotransferase riboflavin synthase, subunit β aspartate aminotransferase conserved hypothetical protein iron-sulfur flavoprotein endopeptidase La iron-sulfur flavoprotein predicted protein adenylosuccinate synthase predicted protein conserved hypothetical protein CODH nickel-insertion accessory protein conserved hypothetical protein sensory transduction histidine kinase alanyl-tRNA synthetase domain protein pneumococcal surface protein 4-carboxymuconolactone decarboxylase hypothetical protein (multidomain) argininosuccinate synthase conserved hypothetical protein conserved hypothetical protein coenzyme A ligase 4-carboxymuconolactone decarboxylase flavoredoxin Flr conserved hypothetical protein conserved hypothetical protein predicted protein mRNA 3-end processing factor orotate phosphoribosyltransferase conserved hypothetical protein HTH DNA-binding protein fructose-bisphosphate aldolase O-acetylhomoserine (thiol)-lyase NifU protein cysteine desulfurase cysteine synthase peptidylprolyl isomerase predicted protein glutamyl-tRNA (Gln) amidotransferase

R S

MA1617 MA1661 MA1672 MA1682 MA1710 MA1712 MA1716 MA1774 MA1778 MA1779 MA1802 MA1805 MA1816 MA1818 MA1819 MA1821 MA1860 MA1862 MA1902 MA1906 MA1919 MA1940 MA1964 MA1967 MA1968 MA1991 MA2014 MA2019 MA2059 MA2106 MA2142 MA2220 MA2238 MA2244 MA2289 MA2295 MA2346 MA2353 MA2386 MA2513 MA2520 MA2558 MA2615 MA2666 MA2715 MA2717 MA2718 MA2720 MA2813 MA2858 MA2862

pI

AC

ME

22,600 13,668 24,722 27,813 15,637 15,434 9,538 23,505 66,225 44,760 12,774 16,445 65,512 15,833 10,475 38,326 24,621 43,885 50,634

4.56 5.08 4.94 5.08 5.23 5.33 4.45 5.75 4.93 5.33 5.26 6.10 6.16 7.64 9.59 6.94 5.92 5.97 4.91

245 309 ND 157 ND 133 253 ND 455 ND 310 254 390 99 ND 504 63 158 333

ND 259 362 56 75 ND 109 228 622 281 544 250 421 76 54 568 ND ND ND

R

28,704

4.59

383

483

O G O H E T C J O E H E H E R R O R

16,162 46,845 58,869 35,319 42,757 15,471 24,639 25,738 27,060 59,087 21,635 42,525 14,953 42,020 54,483 25,601 88,200 29,104 15,676 47,238 18,543 9,901 28,049 22,016 109,795 27,835 50,001 13,452 110,884 44,081 30,567 30,719 49,278 10,710 20,736 35,390 26,910 20,979 46,378 22,231 14,592 30,304 34,596 47,903 14,121 43,327 32,654 28,158 9,995 71,197

5.31 4.48 5.46 4.48 5.00 5.12 5.96 5.73 5.00 5.87 5.29 6.36 5.79 5.27 5.92 5.37 5.66 5.90 5.78 5.89 4.76 6.10 5.47 4.52 5.21 5.43 6.37 5.48 5.44 5.01 6.04 5.81 5.95 4.70 5.76 5.92 5.38 9.50 5.32 5.29 7.63 5.49 5.12 5.90 5.17 6.09 6.47 4.59 9.56 5.28

162 ND 396 349 415 ND ND 492 433 709 55 ND 126 183 381 ND ND 49 210 74 194 ND 400 90 ND ND 223 ND 87 ND 186 391 ND 192 ND 53 35 58 ND 61 ND 366 98 624 ND 162 142 607 ND 182

32 247 311 283 ND 34 66 232 603 414 ND 44 313 ND 91 114 201 ND 486 ND ND 259 143 ND 40 57 157 116 ND 68 ND 154 52 ND 73 ND ND ND 57 47 71 193 76 201 458 484 185 659 50 ND

K T T J O O C J F J R H P E

F F D R T R V E S S H S R P J F R S G E C E E O J

MW

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Table 1. (continued) Mascot scorec loci

gene annotationa

COG codeb

MA2867 MA2868

polyferredoxin heterodisulfide reductase, subunit A/methyl viologen reducing hydrogenase, subunit conserved hypothetical protein formylmethanofuran dehydrogenase, subunit B histone deacetylase hydantoinase/oxoprolinase 2-oxoisovalerate ferredoxin oxidoreductase, R subunit 2-oxoisovalerate ferredoxin oxidoreductase, β subunit transcriptional regulator, Hth-3 family conserved hypothetical protein monomethylamine methyltransferase predicted protein predicted protein anthranilate phosphoribosyltransferase tryptophan synthase, subunit β conserved hypothetical protein conserved hypothetical protein glucose-1-phosphate thymidylyltransferase phosphomannomutase ATPase, AAA family uroporphyrin-III C-methyltransferase coenzyme PQQ synthesis protein E conserved hypothetical protein predicted protein heterodisulfide reductase, subunit C conserved hypothetical protein peptidylprolyl isomerase peptidylprolyl isomerase methanol dehydrogenase regulatory protein (MoxR) carboxymuconolactone decarboxylase hypothetical protein hypothetical protein Sm protein phosphomethylpyrimidine kinase tryptophan synthase, subunit β histidinol dehydrogenase RNase L inhibitor predicted protein thioredoxin ornithine cyclodeaminase CTP synthase phosphoribosylamine-glycine ligase ornithine carbamoyltransferase conserved hypothetical protein excinuclease ABC, subunit B excinuclease ABC, subunit A 2-isopropylmalate synthase glyceraldehyde 3-phosphate dehydrogenase (phosphorylating) dihydroxy-acid dehydratase predicted protein glutamate-ammonia ligase predicted protein conserved hypothetical protein moaA/nifB/pqqE family protein methylenetetrahydrofolate dehydrogenase (NADP+)/methenyltetrahydrofolate cyclohydrolase glycine hydroxymethyltransferase cell division control protein 48 AAA family sensory transduction histidine kinase DNA repair protein small heat shock protein, class I conserved hypothetical protein phosphoglycerate kinase acetate kinase phosphotransacetylase nitrogenase-related protein cobalamin biosynthesis protein conserved hypothetical protein conserved hypothetical protein predicted protein conserved hypothetical protein

C C S C B E C C K Q

MA2875 MA2878 MA2888 MA2889 MA2909 MA2910 MA2914 MA2923 MA2972 MA2982 MA2985 MA2989 MA2991 MA3018 MA3019 MA3022 MA3024 MA3029 MA3033 MA3035 MA3104 MA3113 MA3127 MA3129 MA3136 MA3138 MA3148 MA3152 MA3165 MA3167 MA3195 MA3197 MA3198 MA3201 MA3204 MA3211 MA3212 MA3252 MA3279 MA3309 MA3310 MA3322 MA3323 MA3325 MA3342 MA3345 MA3373 MA3378 MA3382 MA3387 MA3406 MA3482 MA3519 MA3520 MA3527 MA3543 MA3545 MA3589 MA3591 MA3592 MA3606 MA3607 MA3628 MA3631 MA3643 MA3667 MA3673 MA3689 118

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E E S L M G R H R P C K O O R S C C K S R E R O E F F E R L L E G E E O H E O T L O S G C C C S G L R

MW

pI

AC

ME

56,481 88,971

4.46 5.42

249 51

ND ND

11,828 46,558 62,638 61,758 52,198 38,394 21,305 32,545 50,756 34,702 27,077 38,950 44,075 21,500 46,895 44,682 48,045 42,357 27,678 45,496 37,565 15,447 28,473 15,398 17,745 18,172 36,546 12,906 26,245 25,312 8,035 47,283 49,504 46,070 66,201 15,738 10,556 36,236 59,978 47,948 33,867 22,533 77,911 111,301 45,620 36,621 59,107 10,787 57,605 47,358 10,899 40,508 31,635

5.35 6.24 5.75 6.14 5.30 4.87 5.08 4.77 4.92 4.90 4.47 6.19 5.70 4.69 4.82 5.58 4.93 4.96 5.21 6.53 6.17 5.23 8.46 5.19 4.39 4.81 5.15 5.91 8.83 7.52 5.21 5.31 5.27 5.11 5.44 8.99 5.09 4.98 5.41 5.31 5.44 4.85 5.51 5.39 5.21 5.89 5.72 5.14 5.13 5.33 4.16 5.55 5.08

63 ND 522 532 194 170 ND 72 ND 778 537 84 78 579 310 ND 110 ND 64 ND 52 ND ND ND 491 50 238 55 90 93 206 ND 263 151 58 56 339 144 236 111 248 ND 66 46 56 672 457 85 94 ND 230 55 ND

ND 99 ND ND 68 ND 59 ND 85 ND ND 57 ND 510 215 105 ND 47 173 247 ND 55 44 67 94 ND 228 ND 73 ND 42 63 ND 329 ND 29 424 ND 51 ND 52 55 ND ND 59 683 242 62 353 84 ND ND 165

45,111 87,965 73,657 35,253 17,542 21,835 45,528 44,599 35,231 41,005 13,851 14,846 37,130 28,299 44,813

6.57 5.16 5.40 5.29 5.16 4.82 5.98 5.55 4.97 5.36 5.09 4.75 5.57 6.91 6.20

ND 94 49 126 463 186 574 523 276 ND 170 349 157 ND 219

66 133 ND 47 555 ND 129 608 226 75 644 312 ND 61 473

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Proteome of Methanosarcina acetivorans Part I Table 1. (continued)

Mascot scorec loci

gene annotationa

COG codeb

MW

pI

AC

ME

MA3690 MA3692 MA3694 MA3695 MA3715 MA3732 MA3733

translation initiation factor 2, subunit γ DNA-directed RNA polymerase, subunit E conserved hypothetical protein ribosomal protein S24e NH3-dependent NAD+ synthetase F420H2 dehydrogenase, subunit FpoF F420-dependent N5,N10-methylenetetrahydromethanopterin reductase conserved hypothetical protein carboxymuconolactone decarboxylase conserved hypothetical protein glucose-1-phosphate thymidylyltransferase dihydroorotate dehydrogenase, electron-transfer subunit glutamate synthase (NADPH) ketol-acid reductoisomerase acetolactate synthase, small subunit acetolactate synthase, large subunit 2-isopropylmalate synthase integrase predicted protein cellulase prefoldin, subunit β coenzyme F390 synthetase acetolactate synthase, small subunit conserved hypothetical protein carbon monoxide dehydrogenase, subunit R carbon monoxide dehydrogenase, subunit  carbon monoxide dehydrogenase, subunit β carbon monoxide dehydrogenase accessory protein carbon monoxide dehydrogenase, subunit δ archaeal transcription factor E multicatalytic endopeptidase complex, subunit β cleavage and polyadenylation specificity factor cell division protein FtsZ conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein nitrogen regulatory protein P-II ribosomal RNA adenine dimethylase methylcoenzyme M reductase system, component A2 adenylosuccinate lyase lysine 2,3-aminomutase translation initiation factor 5A conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein ABC transporter, ATP-binding protein phosphoribosylaminoimidazolecarboxamide formyltransferase acyl carrier protein synthase acetyl-CoA C-acyltransferase adenylate cyclase seryl-tRNA synthetase translation initiation factor Sui1 conserved hypothetical protein transcriptional regulator, CopG family conserved hypothetical protein universal stress protein glutamate-5-semialdehyde dehydrogenase pyrroline-5-carboxylate reductase peroxiredoxin (alkyl hydroperoxide reductase) prefoldin, subunit R adenylosuccinate synthase H+-transporting ATP synthase, subunit H H+-transporting ATP synthase, subunit E H+-transporting ATP synthase, subunit F H+-transporting ATP synthase, subunit A H+-transporting ATP synthase, subunit B conserved hypothetical protein predicted protein formylmethanofuran dehydrogenase, subunit A formylmethanofuran dehydrogenase, subunit C

J K S J H C C

47,875 21,378 22,120 11,596 42,255 39,221 34,869

6.20 5.23 5.55 5.58 5.56 5.44 6.03

156 230 75 244 392 364 330

110 112 68 664 ND 164 468

S S S M H E E E E E L

9,154 13,109 19,764 26,258 32,916 51,386 36,974 17,673 61,816 52,640 44,865 16,508 37,801 13,427 49,075 15,992 22,120 89,495 18,455 52,792 28,239 47,167 19,787 22,923 72,258 42,407 13,799 37,424 30,901 14,102 37,442 65,758 50,814 48,406 14,271 34,449 19,146 17,934 58,353 59,883 59,735 37,336 41,202 19,867 48,436 11,630 13,945 15,865 31,435 16,370 49,671 27,926 25,203 15,683 46,957 12,252 20,530 10,960 63,965 50,422 64,494 15,925 65,394 28,216

5.27 5.61 4.92 5.04 5.58 5.47 5.02 8.89 5.58 5.38 9.30 9.71 5.56 5.59 5.71 5.46 4.67 5.08 8.98 4.62 5.00 4.58 5.00 5.50 6.58 5.21 4.79 5.78 5.41 5.91 6.23 5.37 5.62 5.80 7.74 6.03 4.86 6.01 4.88 5.43 5.43 5.67 5.24 4.95 5.67 7.62 5.89 5.88 4.94 4.81 5.30 5.13 5.60 4.75 5.27 5.07 5.40 5.35 4.96 5.61 5.51 7.82 5.78 5.25

392 119 92 49 437 291 534 ND ND 167 32 43 241 324 241 104 253 246 204 487 99 158 61 210 ND 427 391 ND 107 ND 118 126 281 49 ND 133 ND ND 66 129 113 153 121 ND 151 ND 171 232 ND 515 171 215 56 203 379 85 ND 51 638 184 73 103 232 ND

ND 154 384 ND 291 237 332 132 94 73 ND ND ND 290 ND 132 ND ND 52 149 ND 199 ND 255 96 760 415 94 344 220 ND ND ND ND 75 ND 121 49 ND ND 107 208 439 163 ND 311 ND 413 158 460 ND 61 204 188 46 ND 215 39 ND 394 268 52 65 363

MA3734 MA3736 MA3742 MA3777 MA3786 MA3787 MA3790 MA3791 MA3792 MA3793 MA3794 MA3810 MA3849 MA3850 MA3853 MA3854 MA3857 MA3860 MA3861 MA3862 MA3863 MA3864 MA3871 MA3873 MA3874 MA3876 MA3884 MA3887 MA3889 MA3916 MA3932 MA3967 MA3971 MA3979 MA3987 MA3992 MA3995 MA3996 MA3997 MA3998 MA4012 MA4041 MA4042 MA4044 MA4048 MA4059 MA4060 MA4075 MA4093 MA4094 MA4100 MA4102 MA4103 MA4110 MA4118 MA4152 MA4155 MA4157 MA4158 MA4159 MA4163 MA4169 MA4175 MA4176

G O H R S C C C D C K O R D S H G E Q R F E J R S S O R F I I F J J R K R T E E O O F C C C C C C C

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Table 1. (continued) Mascot scorec loci

gene annotationa

COG codeb

MW

pI

AC

ME

MA4177 MA4191 MA4195 MA4198 MA4216 MA4217 MA4235 MA4237 MA4240 MA4243 MA4244 MA4258 MA4261 MA4265 MA4268 MA4271 MA4274 MA4275 MA4276 MA4277 MA4329 MA4331 MA4334 MA4344 MA4349 MA4379 MA4382 MA4391

formylmethanofuran dehydrogenase, subunit D conserved hypothetical protein nitrogen fixation protein conserved hypothetical protein glutamate-ammonia ligase conserved hypothetical protein F420-dependent NADP reductase heterodisulfide reductase, subunit B conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein precorrin-8X methylmutase precorrin-4 C11-methyltransferase isocitrate/isopropylmalate dehydrogenase family protein proteasome-activating nucleotidase cell division protein FtsZ ribosomal protein L11p ribosomal protein L1p acidic ribosomal protein P0 homolog ribosomal protein L12p thiamine biosynthesis protein ThiC TATA-binding protein chromosome segregation protein efflux system transcriptional regulator, ArsR family branched chain amino acid aminotransferase methylcobamide:CoM methyltransferase isozyme M CobW protein methanol-5-hydroxybenzimidazolylcobamide co-methyltransferase, isozyme 2 methanol-5-hydroxybenzimidazolylcobamide co-methyltransferase, isozyme 2 conserved hypothetical protein ABC transporter, ATP-binding protein conserved hypothetical protein Hsp60 3-isopropylmalate dehydratase methylenetetrahydromethanopterin dehydrogenase predicted protein conserved hypothetical protein predicted protein UTP-glucose-1-phosphate uridylyltransferase dTDP-glucose 4,6-dehydratase dihydrodipicolinate synthase GMP synthase (glutamine-hydrolyzing) conserved hypothetical protein predicted protein glutamyl-tRNA (Gln) amidotransferase, subunit C glutamyl-tRNA (Gln) amidotransferase, subunit A glutamyl-tRNA (Gln) amidotransferase, subunit B thymidylate synthase methyl coenzyme M reductase, subunit R methyl coenzyme M reductase, subunit γ methyl coenzyme M reductase, subunit C methyl coenzyme M reductase, subunit β deoxyribose-phosphate aldolase conserved hypothetical protein formylmethanofuran dehydrogenase, subunit E triosephosphate isomerase D-arabino 3-hexulose 6-phosphate formaldehyde lyase 2-isopropylmalate synthase conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein conserved hypothetical protein

C R R S E E R C S S S H H E O D J J J J H K D K E H R R

14,165 13,593 36,678 21,203 50,879 39,870 26,823 33,653 25,492 14,117 12,928 26,115 26,390 37,518 49,328 39,610 17,065 23,696 37,201 10,418 47,143 19,985 133,352 13,795 32,391 36,665 50,830 28,253

4.51 5.81 5.78 4.81 5.35 5.71 5.52 5.72 6.01 5.47 6.27 5.04 4.90 5.04 5.52 4.78 5.32 8.87 4.83 3.85 5.75 4.92 5.10 5.64 5.41 5.12 5.75 4.30

ND 112 44 ND 62 ND 396 117 385 ND ND 304 238 111 ND 618 423 78 445 110 364 117 65 252 357 203 ND 732

74 ND 139 168 69 65 139 ND 99 40 110 191 119 324 102 385 376 118 713 312 770 ND 85 ND ND 341 93 563

50,867

4.98

ND

355

39,745 27,719 44,805 58,366 27,585 30,534 10,113 25,196 78,927 35,832 32,715 30,727 34,346 38,194 13,890 10,825 51,367 55,538 26,676 62,435 27,689 19,346 45,393 28,933 41,556 24,846 23,062 42,318 55,943 53,929 23,401 29,439 36,551 31,218 30,573 21,056

4.87 5.79 5.34 5.07 4.70 5.69 5.19 4.80 4.70 5.29 5.66 5.14 5.15 6.34 4.88 4.38 5.56 5.77 4.84 5.14 5.91 5.43 5.34 6.21 5.46 4.93 5.06 5.54 5.23 4.57 4.59 7.79 6.47 6.20 6.19 5.16

172 163 359 64 74 526 223 75 57 50 240 314 ND 466 61 71 ND 386 208 239 645 607 182 222 114 ND 62 371 200 690 149 116 235 236 247 ND

ND ND 529 697 ND 495 85 246 ND 50 ND 116 86 380 ND ND 170 265 ND 382 730 617 894 55 340 138 ND 335 ND 404 ND 56 ND 394 91 96

MA4392 MA4404 MA4406 MA4407 MA4413 MA4415 MA4430 MA4434 MA4436 MA4444 MA4459 MA4464 MA4473 MA4511 MA4517 MA4518 MA4522 MA4523 MA4524 MA4543 MA4546 MA4547 MA4549 MA4550 MA4591 MA4592 MA4602 MA4607 MA4608 MA4615 MA4645 MA4647 MA4648 MA4649 MA4650 MA4651 MA4660

S O O O R C S M M E F S S J J J F H H H H G E C G S E L R R R R R H

a The annotations are the primary annotations listed at http://www.tigr.org/ except for loci MA0750, MA1369, MA2615, and MA3734 that are TIGR annotations. Gene classification code based on Clusters of Orthogonal Groups (COG’s) from http://www.ncbi.nih.gov/. See Table 2 for definition of each letter code. c AC, acetate-grown cells. ME, methanol-grown cells. In the event that a protein was identified in multiple spots in a gel, only the spot with the highest score is presented. A score in bold indicates that at least one MS/MS ion score with p < 0.05 was also obtained for the protein. ND, not detected. b

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Figure 1. Identification of three proteins in a single 2-DE gel spot. (A) MS spectrum of the spot containing three proteins (1, 2, and 3) corresponding to rubrerythrin (MA0639), TATA-binding protein (MA4331) and archaeal transcription factor E (MA3871), respectively. The asterisk indicates the peptides further identified by MS/MS among which three peptides (a, b, and c) are shown in (B) with their MS/MS spectra and detected sequences. Peptides a, b, and c matched to the products of MA4331, MA3871, and MA0639, respectively.

to support growth. Finally, the products of MA3606 and MA3607, with annotations for acetate kinase and phosphotransacetylase, were found in acetate-grown cells (Table 1) which suggests a role in the activation of acetate to acetyl-CoA (step 1, Figure 4) as previously described for M. thermophila.29 (ii) Methanogenesis from Methanol.. The predicted products of MA0455-0456, MA1616-1617 and MA4391-4392 that were detected in methanol-grown cells (Table 1) are paralogs of the 2-subunit methyltransferase, catalyzing the initial reaction in the transfer of the methyl group of methanol to coenzyme M (CoM), as described for Methanosarcina species (step1, Figure 4). The finding that all three paralogs are synthesized in M. acetivorans is consistent with that previously reported for M. thermophila.30 In the pathway for dismutation of methanol to methane, one methyl group is oxidized to CO2 (steps 3-7, Figure 4) to provide the six electrons for reduction of three methyl groups to methane (step 9, Figure 4). The products of MA3733, MA4430, MA1710, and MA0010 were found in methanol-grown M. acetivorans (Table 1) that are predicted to catalyze steps 3-6, consistent with a route for oxidation of the methyl group in M. acetivorans that is identical to that proposed for other Methanosarcina species (Figure 4). The final step in the oxidative branch of the pathway for dismutation of methanol

to methane (step 7, Figure 4) is catalyzed by the 4-subunit, molybdenum-containing, formylmethanofuran (formyl-MF) dehydrogenase (FmdABCD). In the pathway, FmdABCD catalyzes the oxidation of formyl-MF to CO2,31 coupled to the reduction of ferredoxin.32 The genome of M. acetivorans is predicted19 to encode two paralogs of the FmdABCD dehydrogenase within two gene clusters (MA4174-4178 and MA03050309)19 arranged fmdFACDB. Products of MA4175-4177 and MA0306-0308 were detected in methanol-grown M. acetivorans (Table 1). The fmdFACDB gene arrangement in M. acetivorans is the same as that reported for M. barkeri which forms a transcriptional unit,32 consistent with the two gene clusters in M. acetivorans co-transcribed in operons and the synthesis of both 4-subunit FmdABCD paralogs. The results are also consistent with a role for the paralogs in the final step of the oxidative branch for the pathway of methanol conversion to methane that is reported for M. barkeri.20 Both tungsten- (FwdABCD) and molybdenum-containing (FmdABCD) forms of formylmethanofuran dehydrogenases have been characterized from diverse methane-producing Archaea.31-40 The genome of M. acetivorans contains a gene cluster MA0832-0835,19 annotated as fwdCABD, of which the product of MA0832 (FwdC) was found in methanol-grown cells (Table 1). The gene arrangement is consistent with a transcripJournal of Proteome Research • Vol. 4, No. 1, 2005 121

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Figure 2. Identification of the two paralogs of CO dehydrogenase/acetyl-CoA synthase R subunit (CdhA) encoded by MA1016 and MA3860. (A) MS spectrum of the spot containing unresolved CdhA paralogs, with two unique peptides (a and b) each of which matched to only one of the paralogs. (B) The MS/MS spectra and detected sequences of peptide a and b confirming the product of MA3860 and MA1016, respectively. The two peptides differ in only one amino acid residue as underscored in the sequences.

tional unit and apparent synthesis of a tungsten-containing formyl-MF dehydrogenase (FwdABCD). Synthesis of a FwdABCD dehydrogenase in M. acetivorans is in contrast to that reported for M. barkeri,38 suggesting differences between the two species in metal requirements for the enzyme. The genome also contains paired genes (MA2878 and 2879) predicted to encode FwdB and FwdD.19 Although only the product of MA2878 was identified in methanol-grown cells (Table 1), the close proximity of MA2878 to MA2879 is consistent with cotranscription and synthesis of FwdB and FwdD. The genome is not annotated with a second gene encoding either FwdA or FwdC;19 thus, the apparent expression of MA2878-2879 is unexplained unless the FwdCA subunits encoded by MA08320833 are shared with the gene products of MA2878-2879 to produce two functional copies of the tungsten-containing dehydrogenase. Clearly, metal analysis of the enzymes will be necessary to draw any conclusions regarding the synthesis of tungsten- or molybdenum-containing dehydrogenases. Nonetheless, the results are consistent with the synthesis of multiple paralogs of formyl-MF dehydrogenase in M. acetivorans independent of metal content. Gene clusters (MA0306-0309 and MA4175-4178), predicted to encode paralogs of the 4-subunit molybdenum dehydroge122

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nase (FmdABCD), are each clustered with genes predicted to encode a polyferredoxin (MA0305 and MA4174; FmdF)19 that is suggested to accept electrons from the molybdenum dehydrogenase of M. barkeri.32 The FmdF encoded by MA0305 was detected in methanol-grown M. acetivorans (Table 1), a result consistent with a role for the polyferredoxin serving as the electron acceptor for the FmdABCD enzyme encoded by MA0306-0309 in M. acetivorans. (iii) Methanogenesis from both Methanol and Acetate. Both acetate- and methanol-grown M. acetivorans contained the product (MtrH) of MA0269 (Table 1) which is in a gene cluster (MA0269-0276) predicted to encode the 8-subunit (MtrABCDEFGH) methyl-tetrahydromethanopterin (H4MPT):coenzyme M (CoM) methyltransferase19 that is essential in the pathways for methanogenesis from both acetate (step 3, Figure 4) and methanol (step 2, Figure 4), described for Methanosarcina species other than M. acetivorans.20 Although only the MtrH subunit was detected, the arrangement of genes in the MA02690276 cluster19 is consistent with co-transcription in an operon and synthesis of the 8-subunit methyltransferase during growth of M. acetivorans on both substrates. Acetate-grown M. acetivorans also synthesized the product of MA1805 contained in a cluster (MA1804-1806)19 for which MA1804 and MA1805 have

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Proteome of Methanosarcina acetivorans Part I

Table 2. Function Categorization of the 412 Identified Proteins in Methanosarcina acetivorans Based on the Clusters of Orthogonal Groups (COGs) Database sub-function category general function category

code

information

O

storage and processing

identified proteins

description

in AC only

in ME only

in both AC and ME

all

5

4

19

28

T M V D

post-translational modification, protein turnover, chaperones signal transduction mechanisms cell wall/membrane biogenesis defense mechanisms cell cycle control, mitosis and meiosis

2 2 1 2

3 1 0 0

1 1 0 4

6 4 1 6

cellular processes and signaling

J K L B

translation transcription replication, recombination and repair chromatin structure and dynamics

6 6 5 1

12 5 1 0

20 7 4 0

38 18 10 1

metabolism

E C H F G P Q

amino acid transport and metabolism energy production and conversion coenzyme transport and metabolism nucleotide transport and metabolism carbohydrate transport and metabolism inorganic ion transport and metabolism secondary metabolites biosynthesis, transport and catabolism lipid transport and metabolism general function prediction only function unknown not in COGs

12 13 5 4 3 5 2

9 17 7 3 2 0 1

29 23 17 8 12 0 1

50 53 29 15 17 5 4

0 18 14 16

0 14 8 15

3 15 14 10

3 47 36 41

122

102

188

412

poorly characterized

I R S

total

unit. Other than the MA0269-0276 cluster, no other genes encoding subunits MtrB-G are annotated in the M. acetivorans genome19 precluding a rationale for the apparent expression of MA1804-1806. One possible explanation lies in the proposed functions for MtrA and MtrH and the different requirements for steps 2 and 3 (Figure 4) during growth on methanol and acetate. Endergonic step 2 in the direction of methyl-THSPT synthesis is driven by a sodium ion gradient during growth on methanol,42 whereas exergonic step 3 in the direction of methylCoM synthesis is not necessarily dependent on the gradient when metabolizing acetate. MtrH binds methyl-THSPT and transfers the methyl group to MtrA which binds and methylates HS-CoM.43 The remaining subunits MtrB-G function to couple sodium ion translocation driving methyl transfer;43 thus, MtrA and MtrH could function independent of MtrB-G to satisfy step 3 during growth on acetate. The product of MA1805 was only detected in acetate-grown cells (Table 1) in accord with this hypothesis. However, expression of MA1805 and other genes of the MA0269-0276 cluster in methanol-grown cells cannot be ruled out at this juncture. Figure 3. Graphic presentation of function categorization for the 412 genes encoding proteins identified in the proteome of Methanosarcina acetivorans.

50% and 44% identity to MtrH and MtrA from M. thermoautotrophicum.41 The third gene of the cluster (MA1806) is predicted to encode a methyltransferase-related protein (MtrX) of unknown function.19 The genome of M. mazei contains homologues of MA1804-1806, each with greater than 80% identity and clustered in the same arrangement,19 consistent with an important role in the metabolism of Methanosarcina species. Although the product of MA1805 was detected in M. acetivorans (Table 1), conservation of the MA1804-1806 cluster in M. mazei, and the clustered arrangement of the genes in both species,19 is consistent with expression in a transcriptional

Methyl-coenzyme M (CoM) methylreductase catalyzes the demethylation of methyl-CoM in both pathways (steps 4 and 8, Figure 4), as previously described for M. barkeri44,45 and M. thermophila.46 The genome of M. acetivorans is annotated with a gene cluster (MA4546-4550) encoding the 3 subunits (McrABG) of the methylreductase19 for which the products of MA4546 (McrA), MA4547 (McrG), and MA4550 (McrB) were detected in both acetate- and methanol-grown cells (Table 1), consistent with synthesis of a functional 3-subunit enzyme and a role in both pathways. Heterodisulfide reductase from M. barkeri,47,48 M. mazei,49 and M. thermophila50,51 is reported to be essential for methanogenesis from either acetate (step 5, Figure 4) or methanol (step 9, Figure 4). The detection of products for MA3127, MA4237, and MA2868 in either acetate- or methanol-grown Journal of Proteome Research • Vol. 4, No. 1, 2005 123

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the contact point for electron transfer to HdrABC in Methanothermobacter marburgensis.54 The most parsimonious interpretation of this result is that the second putative paralog of HdrABC in M. acetivorans (encoded by MA4236-4237 and MA2868) interacts with a different electron donor than the first HdrABC paralog (encoded by MA3126-3128). Indeed, the product of MA2867 was detected (Table 1) that is predicted to be a polyferredoxin with the potential to function as an electron donor. Furthermore, MA2867 is in close proximity to MA2868 in accord with co-transcription of the gene pair19 suggesting the products have an associated function. Clearly, the results presented here call for further experiments to test the possibility that the HdrABC paralog and polyferredoxin, encoded by MA4236-4237 and MA2867-2868, function during the conversion of either acetate or methanol to methane in M. acetivorans.

Figure 4. Pathways of methanogenesis from methanol or acetate in Methanosarcina species. The figure represents steps in the pathways previously reported for Methanosarcina mazei, Methanosarcina barkeri, and Methanosarcina thermophila. Open arrows and accompanying circled numbers show the steps (1-9) for dismutation of methanol to methane. The solid arrows and accompanying circled numbers show the steps (1-5) for the fermentation of acetate to methane. Substrates and products are shown in large bold font. The four-digit numbers correspond to the gene loci for which products were identified in Methanosarcina acetivorans and are listed in Table 1. Abbreviations: Fdred, ferredoxin (reduced); MF, methanofuran cofactor; H4MPT, tetrahydromethanopterin cofactor; CoA, coenzyme A; CoM, coenzyme M; CoB, coenzyme B; F420H2, coenzyme F420 (reduced).

cells (Table 1) is consistent with the synthesis of two paralogs of the soluble, flavin-containing, three-subunit (HdrABC) enzyme52,53 in M. acetivorans. Although only the product of MA3127 (HdrC) was detected, the gene overlaps with both MA3126 and MA3128 predicted to encode HdrB and HdrA,19 consistent with coexpression of the MA3126-3128 cluster and synthesis of the first of two potential HdrABC paralogs. The gene pair MA4236 and MA4237 is annotated as encoding HdrC and HdrB for which only the MA4236 gene product was detected (Table 1). The close proximity of MA4236 and MA423719 is consistent with coexpression and that two (HdrBC) of the three subunits of a second HdrABC paralog are synthesized. A predicted third subunit (HdrA) for the second HdrABC paralog was identified (Table 1), although it is encoded by MA2868 that is remote from any of the hdr genes on the chromosome.19 Nonetheless, the results are consistent with the synthesis of a second HdrABC paralog. Notably, MA2868 is annotated as hdrA fused with a gene predicted to encode MvhD (Table 1) that is 124

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(iv) Redundancy of Enzymes. A remarkable feature of the genomic sequences of M. mazei55 and M. acetivorans4 is the extensive gene redundancy which has raised questions regarding the expression of these genes and their physiological significance. Most striking is the redundancy of genes predicted to encode enzymes that function in methanogenesis pathways. The results presented here show a pattern of redundancy in the synthesis of enzymes essential for both pathways of methanogenesis from either acetate or methanol in M. acetivorans. The physiological basis for this redundancy is untested, although several possibilities can be envisioned. One possibility is that the paralogs have specificity for a pathway suggested by apparent differences in subunit composition for which paralogs of the heterodisulfide reductase is an example. Another possibility is that paralogs are each encoded by independently regulated operons to provide a mechanism for adjusting levels of the enzyme. Indeed, the accompanying report12 suggests differential regulation of the three operons encoding the methyltransferase that functions in step 1 (Figure 4) of the pathway for methanogenesis from methanol. Synthesis of paralogs could also be an adaptation to the specific environment of the species, as illustrated by the apparent synthesis of a tungsten-containing formyl-MF dehydrogenase in M. acetivorans, whereas M. barkeri only synthesizes the molybdenumcontaining enzyme. Finally, it cannot be ruled out that gene duplication and loss occurs regularly and has no selective pressure. Conservation of Energy. The gene cluster MA0659-0664 is predicted19 to encode the six subunits of a sodium-transporting NADH:ubiquinone oxidoreductase (Nqr) described for marine microbes,4,56 although not previously described for any methaneproducing species. Although only the product of MA0659 was detected in acetate-grown M. acetivorans (Table 1), the arrangement of genes in the MA0659-0664 cluster19 is in accord with co-transcription and synthesis of the 6-subunit oxidoreductase. Indeed, transcriptional mapping confirms that the cluster forms a transcription unit (unpublished results). Nqr is an entry for electrons into the respiratory chain of marine microbes which generates a sodium motive force for metabolic work and possibly ATP synthesis.57 It has been proposed that a quinone-like electron carrier, methanophenazine, is the electron donor to the Hdr in acetate-grown Methanosarcina species.50 Thus, a potential function for Nqr in M. acetivorans is to link electron transport between reduced ferredoxin and methanophenazine with generation of an ion motive force. Genes encoding the Nqr are absent in the genome of M. mazei,55 and the Ech hydrogenase complex is postulated to generate an ion motive force coupled with the oxidation of

Proteome of Methanosarcina acetivorans Part I

ferredoxin and reduction of methanophenazine.58 Conversely, the genome of M. acetivorans does not encode a functional Ech hydrogenase,4 consistent with Nqr replacing the Ech hydrogenase. Regulation. Relatively little is known concerning transcriptional regulation in the methanogenic Archaea. Methanosarcina species are the most metabolically diverse among the Archaea, and show extensive regulation in response to changes in growth conditions29,59-64 which affords opportunities for understanding principles of gene regulation in Methanosarcina and the Archaea domain. The basal transcription apparatus in the Archaea resembles that of the Eucarya domain involving a TATA-box binding protein (TBP) and a transcription factor B which complexes with a eucaryal-like RNA polymerase.65,66 Notwithstanding the commonalities between the basal transcription machinery of the Archaea and Eucarya domains, the genome of M. acetivorans harbors an abundance of genes encoding transcription regulators also present in the Bacteria domain4 which prompts questions concerning their role in the physiology of M. acetivorans and how interactions between the bacterial regulators and archaeal basal transcription apparatus fulfill those roles. Several transcriptional regulators (MA3916, MA4344, MA0750, MA4404, MA2914, and MA2615) were found to be synthesized in either acetate- or methanol-grown cells (Table 1), providing an important first step in elucidating details of gene regulation in Methanosarcina species and the Archaea. The predicted product of MA3916, nitrogen regulatory protein P-II (GlnK), was found in methanol-grown cells (Table 1). GlnK has been characterized from M. mazei and shown to regulate ammonium assimilation.67 As is the case in M. mazei, glnK in M. acetivorans is adjacent to a gene encoding an ammonium transporter,19 consistent with the regulatory function for GlnK. M. acetivorans was cultured with ammonium as the nitrogen source; however, the expression of glnK in M. mazei is repressed under these growth conditions suggesting either a different mechanism for regulation or possibly a different regulatory role for GlnK in M. acetivorans. There is a paucity of understanding concerning toxic metal metabolism or resistance by the methane-producing Archaea. Both methanol- and acetate-grown M. acetivorans contained a putative efflux system transcriptional regulator of the ArsR family of metalloregulatory proteins encoded by MA4344 and MA0750 (Table 1) that in members of the Bacteria domain respond to a variety of metals including As(III), Sb(III), Cd(II), and Zn(II). In E. coli, arsR is the first gene of the ars operon which confers low level resistance to arsenicals and antimonials.68 Although not detected in M. acetivorans, MA4345 overlaps with MA434419 and is predicted to encode a putative transporter consistent with co-transcription of MA4344-4345 and a potential role for the MA4345 product in conferring metal resistance through efflux. Methanosarcina species grown with acetate or methanol require an abundance of metalloproteins containing a variety of metals (Zn, Ni, Co, Mo, and Fe) of which the unbound forms could be toxic and require exquisite control of the cytoplasmic concentrations as previously proposed for E. coli.69 Although annotated as a conserved hypothetical protein (Table 1), the sequence of MA4404 has 34% identity to the recently described novel regulatory protein NrpR shown to repress nif (nitrogen fixation) and glnA (glutamine synthetase) expression in methane-producing Methanococcus maripaludis.70 The gene product of MA4404 was found in acetate-grown M. acetivorans (Table 1) cultured with ammonium as the

research articles nitrogen source, consistent with the proposed role in M. maripaludis, although other regulatory roles cannot be ruled out. MA4404 is closely aligned with MA4405,19 annotated as a metallo-β-lactamase superfamily protein, consistent with coexpression of the gene pair. No evidence was obtained for the presence of a NpR paralog,70 the predicted product of MA0822, in M. acetivorans grown with either acetate or methanol. The predicted product of MA2914 is a putative transcriptional regulator of the Hth-3 family and was found to be synthesized in methanol-grown cells (Table 1). MA2914 is the first of three tightly clustered genes19 where MA2913 encodes a predicted protein and MA2912 is annotated as encoding an acetyl-CoA synthetase catalyzing the ATP-dependent synthesis of acetyl-CoA from acetate. Although the product of MA2912 was not detected, clustering of the three genes is consistent with coexpression and synthesis of the acetyl-CoA synthase during growth on methanol. Acetyl-CoA is an essential precursor for synthesis of cell carbon, and the only other pathway for acetate conversion to acetyl-CoA is dependent on acetate kinase and phosphotransacetylase, both of which are repressed during growth of M. acetivorans on methanol,12 as was also shown for M. thermophila.29 Finally, another putative helixturn-helix DNA binding protein, encoded by MA2615, was found to be present in both acetate- and methanol-grown cells (Table 1), although MA2615 is not clustered with genes that would indicate a potential function. Protein Folding and Maturation. The presence of genes encoding both the group I and II chaperonins in Methanosarcina species4,55 raises questions regarding the division of function for these groups. It is reported that both groups are synthesized in methanol-grown M. mazei.71 The finding that the group I chaperonins GroEL (MA0631), GroES (MA0630) and Hsp60 paralogs (MA0086, MA1682, and MA4413) are synthesized in methanol-grown cells (Table 1) of M. acetivorans is consistent with the results obtained for M. mazei. Each of these proteins were also found to be synthesized in acetate-grown M. acetivorans (Table 1), extending the range of growth conditions under which the group I chaperonins are synthesized in Methanosarcina species. Although no evidence was obtained for synthesis of the group II thermosome, the finding that the R and β subunits of prefoldin (MA4110 and MA3850) are present in methanol-grown cells (Table 1) is consistent with synthesis of an accompanying thermosome in M. acetivorans as described for M. mazei.71 The genome of M. acetivorans is annotated with five genes predicted to encode peptidyl-prolyl isomerase paralogs.19 The products of four of these genes (MA1661, 2813, 3136, and 3138) were detected in either acetate- or methanol-grown cells (Table 1). Although the peptidyl-prolyl isomerase, predicted to be encoded by MA3137, was not detected, the gene order in the MA3136-3138 cluster is consistent with a transcriptional unit and with synthesis of all 5 paralogs. Very little is known regarding the existence or specific function of peptidyl-prolyl isomerases in the Archaea domain.72 The only reported characterization of this enzyme for methane-producing species is a FK506 binding protein from Methanococcus thermolithotrophicus73 that exhibits both peptidyl-prolyl isomerase and chaperonin-like activity. The finding that at least four paralogs of peptidyl-prolyl isomerase are synthesized in M. acetivorans suggests an important role for this enzyme. Interestingly, a peptidyl prolyl cis/trans isomerase is reported to be synthesized in different amounts in Methanococcoides burtonii dependent on the growth temperature.11 Journal of Proteome Research • Vol. 4, No. 1, 2005 125

research articles The gene product of one (MA3148) of four moxR homologues (MA0254, 2200, 2842, and 3148) annotated in the M. acetivorans genome19 was detected in both acetate- and methanol-grown cells (Table 1). Members of the MoxR family are widely distributed in the Bacteria and Archaea domains, yet none of the proteins have been characterized.74 Although annotated as a methanol dehydrogenase regulatory protein (Table 1), MoxR and other members of the family apparently function as chaperonins in the assembly of complexes.74 The prototypic MoxR is involved in the biogenesis of the methanol dehydrogenase complex of methanol-utilizing aerobes,74 although a specific function has not been determined. The finding that MoxR is synthesized in M. acetivorans, grown with either substrate, suggests that it has a more general role rather than the biogenesis of proteins specific to methanol utilization. Small nuclear ribonucleoproteins, which function in RNA processing of Eucarya, contain Sm and Sm-like (Lsm) proteins that are also encoded in the genomes of Archaea,75 including M. acetivorans (MA1413 and MA3195),19 for which the MA3195 gene product was detected (Table 1). The function of Sm/Lsm proteins in the Archaea is unknown; however, in several species, the genes encoding LsmR are immediately upstream of L37 encoding a ribosomal protein, leading to the speculation that Sm/Lsm proteins fulfill ribosomal functions or are involved in ribosome biogenesis.75 MA3195, encoding the expressed LsmR in M. acetivorans, is upstream of a gene predicted to encode L37,19 consistent with functions postulated for the Archaea. Stress. The products of several genes (MA0639, 4103, 1574, and 3212) were identified in M. acetivorans (Table 1) that are predicted to encode proteins which function in the oxidative stress response. The predicted products of MA0639 and MA4103 are rubrerythrin and peroxiredoxin (alkyl hydroperoxide reductase) which catalyze the reduction of hydrogen peroxide to water. M. acetivorans also contained (Table 1) a putative superoxide dismutase (MA1574) and two putative thioredoxins (MA3212 and MA1369). Thioredoxins function in the oxidative stress response of aerobes by donating electrons to peroxidases such as peroxiredoxin76 and have been characterized from Methanococcus jannaschii77 and Methanothermobacter thermoautotrophicus (Methanobacterium thermoautotrophicum),78 both methane-producing species from the Archaea domain. The deduced sequence of MA1369 has 35% identity to the M. jannaschii thioredoxin that is proposed to reduce disulfide bonds formed during oxidative stress.77 Although proliferating in anaerobic habitats, methanogenic Archaea encounter oxygen and, similar to anaerobes from the Bacteria domain,79 are likely to have evolved complex mechanisms for responding to oxidative stress; indeed, Methanosarcina species are the most tolerant of the methanogenic Archaea toward oxygen, surviving long periods of exposure to air.80 The results presented here indicate that M. acetivorans synthesizes a diverse inventory of proteins for response to oxidative stress. Conserved Proteins. A large number of proteins annotated as either “conserved hypothetical” or “predicted” were identified (Table 1), establishing these species as bona fide proteins synthesized in M. acetivorans and identifies them as potential targets of investigation to determine function. None of the putative genes encoding these proteins were clustered with genes predicted to encode proteins with known functions that could suggest a potential function, except where noted elsewhere in the text. The product of MA3019 found in M. acetivorans (Table 1) is annotated as conserved; however, the 126

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protein was recently overproduced in E. coli and shown to be a DNA replication A protein.6

Conclusions This, the first proteomic analysis of a Methanosarcina species, has revealed several novel features of the genus. A distinguishing trait of the genomes of Methanosarcina species is a surprising number of apparently redundant genes4,55 for which the products of several involved in the pathways for methanogenesis were found in M. acetivorans, suggesting essential functions for the gene redundancy. Given the overlap on 2D gel spots for the protein products of these redundant genes, MALDI-TOF-TOF MS analysis was critical to the identification of these products. A wealth of genes were found to be expressed that encode enzymes and proteins with the potential to respond to oxidative stress, a result which explains the unique ability of Methanosarcina species to survive in the presence of air. Evidence was obtained for synthesis of a sodium-transporting oxidoreductase with potential for participation in conservation of energy during growth with acetate. Several regulatory proteins were found that commonly function in the Bacteria domain suggesting novel and complex interactions for coordination with the Eucarya-like basal transcription apparatus of M. acetivorans. In summary, the results of this analysis has furthered the biological understanding of M. acetivorans which provides the foundation for experimentation toward improvement of the rate and reliability of methanogenesis. In Part II,12 proteins have been identified that are present in different amounts between acetate- and methanolgrown M. acetivorans which builds on the results presented here to further define the biology of Methanosarcina species with respect to the specific growth substrate.

Acknowledgment. This work was supported by NSF Grant No. MCB-0110762 to J.G.F. with a subcontract to B.L.K. Contribution #844 from the Barnett Institute. References (1) Schlesinger, W. H., The global carbon cycle. In Biogeochemistry, Academic Press: San Diego, 2000; pp 308-321. (2) Sowers, K. R.; Baron, S. F.; Ferry, J. G., Methanosarcina acetivorans sp. nov., an acetotrophic methane-producing bacterium isolated from marine sediments. Appl. Environ. Microbiol. 1984, 47, 971978. (3) Metcalf, W. W.; Zhang, J. K.; Apolinario, E.; Sowers, K. R.; Wolfe, R. S., A genetic system for Archaea of the genus Methanosarcina. Liposome-mediated transformation and construction of shuttle vectors. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2626-2631. (4) Galagan, J. E.; Nusbaum, C.; Roy, A.; Endrizzi, M. G.; Macdonald, P.; FitzHugh, W.; Calvo, S.; Engels, R.; Smirnov, S.; Atnoor, D.; Brown, A.; Allen, N.; Naylor, J.; Stange-Thomann, N.; DeArellano, K.; Johnson, R.; Linton, L.; McEwan, P.; McKernan, K.; Talamas, J.; Tirrell, A.; Ye, W.; Zimmer, A.; Barber, R. D.; Cann, I.; Graham, D. E.; Grahame, D. A.; Guss, A. M.; Hedderich, R.; Ingram-Smith, C.; Kuettner, H. C.; Krzycki, J. A.; Leigh, J. A.; Li, W.; Liu, J.; Mukhopadhyay, B.; Reeve, J. N.; Smith, K.; Springer, T. A.; Umayam, L. A.; White, O.; White, R. H.; de Macario, E. C.; Ferry, J. G.; Jarrell, K. F.; Jing, H.; Macario, A. J.; Paulsen, I.; Pritchett, M.; Sowers, K. R.; Swanson, R. V.; Zinder, S. H.; Lander, E.; Metcalf, W. W.; Birren, B., The genome of M. acetivorans reveals extensive metabolic and physiological diversity. Genome Res. 2002, 12, (4), 532-542. (5) Freitas, T. A.; Hou, S.; Dioum, E. M.; Saito, J. A.; Newhouse, J.; Gonzalez, G.; Gilles-Gonzalez, M. A.; Alam, M., Ancestral hemoglobins in Archaea. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, (17), 6675-6680. (6) Robbins, J. B.; Murphy, M. C.; White, B. A.; Mackie, R. I.; Ha, T.; Cann, I. K., Functional analysis of multiple single-stranded DNAbinding proteins from Methanosarcina acetivorans and their effects on DNA synthesis by DNA polymerase BI. J. Biol. Chem. 2004, 279, (8), 6315-6326.

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