Cold Adaptation of the Antarctic Archaeon, Methanococcoides burtonii

Synopsis. Isotope coded affinity tag (ICAT) chromatography and liquid chromatography−mass spectrometry were used to identify differentially expresse...
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Cold Adaptation of the Antarctic Archaeon, Methanococcoides burtonii Assessed by Proteomics Using ICAT Amber Goodchild,† Mark Raftery,‡ Neil F. W. Saunders,† Michael Guilhaus,‡ and Ricardo Cavicchioli*,† School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, 2052, NSW, Australia and Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Sydney, 2052, NSW, Australia Received December 16, 2004

Using isotope coded affinity tag (ICAT) chromatography and liquid chromatography-mass spectrometry, 163 proteins were identified from the cold-adapted archaeon, Methanococcoides burtonii. 14 proteins were differentially expressed during growth at 4 °C and 23 °C. Knowledge of protein abundance, protein identity and gene arrangement was used to determine mechanisms of cold adaptation. Growth temperature was found to affect proteins involved in energy generation and biosynthesis linked to methanogenesis, membrane transport, transcription and protein folding, as well as affecting the expression of two hypothetical proteins. Pooling the data from this ICAT study with data from a previous two-dimensional gel electrophoresis study highlighted consistencies and differences between the two methods, and led us to conclude that the two approaches were generally complementary. This is the first report of ICAT applied to Archaea, or for the study of cold adaptation in any organism. Keywords: Archaea • methanogen • proteome • cold adaptation • psychrophile • ICAT

Introduction Isotope coded affinity tag (ICAT) labeling of proteins coupled with tandem mass-spectrometry (MS/MS)1 has proven to be an effective technology for identifying and quantifying differentially expressed proteins. It represents one of several recently developed, stable-isotope labeling methods for examining differential expression.2 ICAT involves the labeling of cysteine residues in proteins with 12C or 13C. Following affinity chromatography and MS/MS, the relative abundance of the isotopes associated with peptides from the two labeled samples provides an accurate quantification and identification of differentially expressed proteins. A strength of the ICAT-method is the reduction in sample complexity prior to MS-analysis, thereby facilitating the detection of less abundant peptides. An obvious limitation of the method is the inability to detect peptides if they do not contain cysteine residues. Irrespective of the inherent limitation, the ability to perform high-throughput, liquid chromatography (LC)-based proteomics has resulted in ICAT being used to identify differentially expressed proteins from Pseudomonas aeruginosa,3 rat cells,4 prostate cells,5 and Trypanosoma cruzi.6 The method has also proven useful for quantifying protein abundance in macromolecular complexes,7 and for identifying low abundance8 and membrane bound proteins.9 Methanococcoides burtonii is a microorganism that has adapted to life in Antarctica where it resides in Ace Lake at * To whom correspondence should be addressed. Tel. +61-2-93853516. Fax. +61-2-93852742. E-mail. [email protected] † School of Biotechnology and Biomolecular Sciences. ‡ Bioanalytical Mass Spectrometry Facility. 10.1021/pr049760p CCC: $30.25

 2005 American Chemical Society

temperatures that remain permanently 1-2 °C.10 M. burtonii is a member of the Archaea; a domain of life distinct from the Bacteria and Eucarya.11 While Archaea are prevalent in the Earth’s cold biosphere, few cold adapted archaea have been isolated. Most studies of archaeal cold adaptation have been performed on M. burtonii, including studies of protein structure,12-15 intracellular solutes,15 tRNA,16 lipids,17 gene regulation,18 and comparative genomics.19 Recently, we developed two-dimensional electrophoresis (2DE) coupled to MS/MS to identify proteins differentially expressed in M. burtonii during growth at 4 °C and 23 °C.20 In addition, LC/LC-MS/MS methods were developed and 528 proteins were identified from cultures growing at 4 °C.21 The large number of proteins identified by LC/LC-MS/MS enabled a view of the biology of cold adaptation of M. burtonii to be advanced,21 including the examination of the role of 135 hypothetical proteins.22 Proteins from cold-adapted organisms (psychrophiles) tend to be thermolabile in comparison to proteins from organisms growing at high temperature.23,24 As a result, proteomic studies of archaea have tended to be performed on the hyperthermophile, Methanocaldococcus jannaschii; a fact which also stems from M. jannaschii being the first archaeon to have its genome completely sequenced.25 Our ability to establish high-throughput analysis of proteins from M. burtonii21 has provided the basis for developing ICAT. In the present study, we adopted the same temperatures (4 °C and 23 °C) and growth conditions that were used for the 2DE study,20 enabling a direct comparison of the two methods. The use of ICAT expanded the number of proteins known to be expressed and differentially expressed Journal of Proteome Research 2005, 4, 473-480

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research articles in M. burtonii, enabling us to generate a more comprehensive and accurate view of cold adaptation. The ability to apply ICAT to proteins from M. burtonii also demonstrated that the technology may be generally applicable to proteomic studies of psychrophiles, and other members of the Archaea.

Experimental Procedures Organisms and Culture Conditions. M. burtonii was grown at 4 °C and harvested as previously described.20 Sample Preparation, ICAT Labeling, Liquid Chromatography, and Mass Spectrometry. Protein extraction was performed as previously described.21 The lyophilised ICAT reagents (Applied Biosystems) were resuspended in 20 µL of acetonitrile. The 4 °C protein sample was transferred to a vial containing the light (12C) reagent and the 23 °C sample transferred to a vial containing the heavy (13C) reagent, and incubated at 37 °C for 2 h. The two samples containing the labeled 4 °C and 23 °C proteins were mixed and digested with 1 µg trypsin (Applied Biosystems) at 37 °C for 16 h. 200 µg of digested protein was mixed with 4 mL of cation exchange loading buffer (all buffers and columns from Applied Biosystems) and loaded at a rate of 1 drop s-1 onto a cation exchange cartridge conditioned with 2 mL of buffer. The column was washed with a further 2 mL of buffer and the peptides eluted at a rate of 1 drop s-1 using 0.5 mL of cation exchange elution buffer. The eluate was neutralized with 0.5 mL of affinity loading buffer and loaded at a rate of 1 drop s-1 onto an avidin column which had been prewashed with 2 mL of affinity elution buffer and 2 mL affinity loading buffer. The nonlabeled material was removed from the column by injecting 0.5 mL affinity loading buffer, 1 mL affinity washing buffer 1, 1 mL affinity washing buffer 2 and 1 mL of Milli-Q water. Peptides were eluted at a rate of 1 drop s-1 by injecting 0.8 mL of affinity elution buffer. The eluate was dried in vacuuo, mixed with a 95:5 solution of Cleaving Reagent A (Applied Biosystems):Cleaving Reagent B (Applied Biosystems), incubated at 37 °C for 2 h and dried in vacuuo. Separation of peptides using LC and MS was performed as previously described.21 MS/MS Analysis. Collision induced dissociation (CID) spectra generated from LC/LC-MS/MS were analyzed using Mascot (Matrix Science), AnalystQS software (Applied Biosystems) or ProICAT software (Applied Biosystems). The Mascot search parameters were: trypsin digestion allowing up to 1 missed cleavage, variable modifications +16 methionine (oxidation), fixed modifications +227.14 and +236.15 cysteine (light and heavy reagents respectively), peptide tolerance of 1.0 Da and MS/MS tolerance of 0.8 Da. All MS/MS spectra were manually verified. The ProICAT search parameters were: peptide tolerance 0.5 Da, MS/MS tolerance 0.25 and confidence >50. All searches were performed on a local database of M. burtonii translated sequences.21 Searches with a high confidence (>50) and high score (>20) were judged to provide a positive identification. The MS/MS spectra for all peptides satisfying these criteria was confirmed by visual inspection. The peptides were quantified using ProICAT, or AnalystQS by computing the MS-derived chromatographic peak area for each peptide doublet. The ProICAT software automatically computes the areas and 12C:13C averaged ratios for pairs of peaks detected in the TOF MS scan. A 2-fold change in the protein abundance ratios (12C:13C) and the presence of the peptide in all 3 labeling experiments was set as the threshold for a significant change. Previous studies have reported changes as significant if peptide abundance ratios were between 0.5 and 474

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0.67, or 1.5 and 2.0.26 The variation in the relative abundance of differentially expressed peptides between the 3 labeling experiments was analyzed by computing the standard deviation and by performing a t-test (P < 0.05). Computational Analyses. Draft genome data are based on the JGI assembly of 12-Nov-03 and the ORNL Genome Channel analysis of 11-Dec-03 (http://maple.lsd.ornl.gov/microbial/ mbur/). Gene prediction and analysis procedures were performed as previously described (refs 20-22). Putative operon structure was predicted using the FGENESB application at the Softberry website (http://www.softberry.com).

Results and Discussion M. burtonii ICAT Proteome. Protein extracts from M. burtonii cultures grown at 4 °C and 23 °C were labeled with the ICAT reagent and digested with trypsin. ICAT-labeled peptides were isolated using affinity chromatography, separated by LC/LC and analyzed by MS/MS. 163 proteins were identified (Table S1), including 30 proteins (Table 1) not previously identified by LC- and LC/LC-MS/MS from unlabeled extracts.21 To assess the coverage of ICAT-labeled proteins, a plot of predicted pI vs MW was generated for each protein, and compared with the equivalent data from the 528 previously identified unlabeled proteins21 (Figure 1). The predicted pI and MW of the ICAT-labeled proteins ranged from 3.9 to 11.9 and 5 kDa to 112 kDa, respectively. Overall the distribution patterns were similar for the two methods, although the dynamic range (pI and MW) for the ICAT-labeled proteins was marginally lower. This difference may be explained by chance resulting from the smaller number of proteins identified by ICAT-labeling (163 vs 528). It is noteworthy that similar differences in the number of proteins identified between ICAT-labeled and unlabeled extracts has previously been reported in studies using Saccharomyces cerevisae.9,26 Codon usage can be a useful indicator of gene expression levels and we have previously correlated codon usage with protein abundance using correspondence analysis.21 In the study, proteins that were predicted to be more abundant in M. burtonii distributed to the right-hand side of a codon usage plot.21 Analysis of the codon usage for each of the genes encoding the proteins identified from the present ICAT-labeling experiments revealed proteins distributed throughout the plot with a bias toward the right-hand side (Figure 2). This indicated that the M. burtonii proteins identified using ICAT-labeling tended to be relatively abundant proteins. To verify this visual impression, the median value for the codon usage of all of the genes in the genome was calculated (represented by the dotted vertical line in Figure 2), and used to quantify the proportion of genes that distributed on either side of the median. 89% of the ICAT-labeled proteins fell to the right-hand side, compared with only 77% of those identified by LC- and LC/LC-MS/MS analysis of unlabeled M. burtonii proteins. The decreased complexity resulting from ICAT sampling has been reported to aid in the detection of low abundance proteins.8,27,28 The tendency to detect the more abundant proteins in M. burtonii extracts may indicate that the low abundance proteins are not labeled or purified as effectively. We previously found that repeat LC/LC-MS/MS runs of unlabeled peptides provided an increased number of newly identified proteins, although this occurred with a decreasing return for each repeat (e.g., 278 for the 1st run, 84 for the 2nd, 47 for the 3rd, 38 for the 4th).21 To some degree increasing the

ICAT proteomics of Methanococcoides burtonii

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Table 1. Proteins Not Previously Detected in the Expressed Proteome of M. burtonii contig•genea

67•1056 70•2293 58•650 54•440 57•625 70•2311 70•2212 67•1513 61•857

69•2183 65•1225 53•401 44•80 68•1788 69•2112 61•909 55•467 63•1062 70•2732 70•2326 67•1516 67•1623 56•551 59•749 66•1348 70•2575 68•1726 48•190 68•1890 52•339

functional category

DNA replication and processing Putative DNA-binding protein RNA synthesis and processing Archeosine tRNA-ribosyltransferase Pseudouridine synthase Signal transduction Sensor histidine kinase Motility Flagella accessory protein I Protein synthesis and processing LSU ribosomal protein L37E LSU ribosomal protein L24E Aspartate aminotransferase Phenylalanyl-tRNA synthetase Post-translational 177408, protein degradation and chaperones PmbA protein Phosphoesterase Cell envelope Glucose-1-phosphate thymidyltransferase Long-chain-fatty-acid-CoA ligase Methanogenesis Monomethylamine methyltransferase MtmB Energy production and conversion NADP oxidoreductase Na+-transporting NADH:ubiquinone oxidoreductase subunit 6 Ferredoxin Fe-S binding protein Fe-S flavoprotein Carbon fixation and carbohydrate metabolism Pyruvate synthase ∆ subunit Predicted sugar kinase Carboxymuconolactone dehydrogenase Nucleotide metabolism Phosphoribosyltransferase Riboucleoside triphosphate reductase activating enzyme Dihydroorotate dehydrogenase Amino acid metabolism Urease accessory protein Nitrogenase associated protein E Coenzyme metabolism Anthranilate synthase Coenzyme PPQ synthesis protein Quinolinate synthetase A

a contig•gene numbers are from the 11Dec03 release of the M. burtonii genome sequence annotation.

number of replicates may enhance the number of proteins identified using ICAT. We calculated the proportion of proteins predicted to encode at least one cysteine residue from the M. burtonii draft genome, and all complete archaeal genome sequences. The proportion ranged from 87% for Methanopyrus kandleri to 58% for Nanoarchaeum equitans, with M. burtonii having 80%. All methanogens and Archaeoglobus fulgidus (7 genomes) had 80% or greater, while all other archaea (11 genomes) had 69% or less. Not withstanding the bias toward identifying the more abundant proteins from M. burtonii, the reasonable proportion of predicted cysteine containing proteins in Archaea indicates that ICAT technology may be generally useful for proteomic studies in Archaea; particularly for the methanogens with a higher cysteine content. Differential Expression. To evaluate the ICAT method for differential expression of M. burtonii proteins, a number of variations were trialed and controls implemented. The relative abundance of peptides was quantitated using ProICAT soft-

Figure 1. Simulated 2D gel of the M. burtonii proteome. Predicted MW and pI for proteins identified in this study using ICAT (black, closed circles) and 528 proteins identified from unlabeled samples (grey, open circles) in a previous study.21

Figure 2. Correspondence analysis of codon usage frequency variation for genes from M. burtonii. Genes corresponding to proteins identified in this study using ICAT (black, closed circles) and 528 proteins identified from unlabeled samples (grey, open circles) in a previous study.21 The median value for the codon usage of all of the genes in the genome is represented by the dotted vertical line.

ware, or by computing the MS-derived chromatographic peak area for each peptide doublet using AnalystQS. The output from the two methods was compared in a scatter plot (Figure 3a). The close correspondence of the data indicated that for quantitation purposes the two methods produced equivalent results. However, an advantage of the ProICAT software was the reduced requirement for manual processing. Comparing the relative abundances of the peptides from the same labeling experiment following two separate LC/LC-MS/MS runs (Figure 3b), also demonstrated that the quantitation of peptides between runs was reproducible. To assess the consistency of labeling, the same sample was independently labeled twice, and the data compared (Figure 3c). All of the same peptides were not identified because they were not always selected for MS/MS. However, the relative abundance of the peptides that were identified from both experiments was comparable (Figure 3c). Furthermore, of the 163 proteins identified 48 were identified from 2 or more peptides. The standard deviation of the range of the relative abundances was 1.5-fold and 1 more increased >2-fold. At 23 °C, Journal of Proteome Research • Vol. 4, No. 2, 2005 475

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Figure 3. Analysis of reproducibility of labeling and quantitation of M. burtonii proteins using ICAT. (A) Relative abundance of peptides analyzed in a single LC/LC-MS/MS run calculated using ProICAT and AnalystQS. (B) Relative abundance of two separate LC/LC-MS/MS runs of the same sample. (C) Relative abundance of a single sample following two independent rounds of ICAT labeling, and LC/LC-MS/MS analysis.

the abundance of 6 proteins increased >1.5-fold and an additional 6 by >1.5-fold. An example of MS data is shown for 476

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the chaperonin protein, Cpn60, illustrating the mass spectra for the labeled doublets that were differentially expressed (Figure 4a), and the tandem mass spectra for peptide identification (Figure 4b). It is noteworthy that all mass spectra for all proteins identified and quantified in this study were manually verified and a protein was only considered to be differentially expressed if it was at least 1.5-fold more abundant26 at one of the growth temperatures in at least two replicates. Using 2DE, 26% of spots from triplicate gels at both growth temperatures were found to have differential spot intensities of 2-fold or more using the Z3 software program.20 The standard deviation of these spot intensites ranged from 5% to 30%. Twenty one of these spots were more intense at 4 °C and 33 more intense at 23 °C from a total of 209 spots that appeared in all 3 gel replicates.20 From these gels, 43 spots corresponding to 33 proteins were identified. The total number of unique proteins, differentially expressed by at least 2-fold (16%) in the 2DE study was 4-fold higher than the equivalent number identified by ICAT in the present study. The lower proportion is likely to reflect the stringent criteria applied to the ICAT data which required that the relative abundance of an ICAT-labeled peptide needed to be greater than 2-fold in all three labeling experiments. As a result, peptides that were not selected for CID in one of the three runs were excluded from our list. Although this approach reduced the number of differentially expressed proteins that could be identified, it provided a high level of confidence for those that were identified. A number of studies have used one round of ICAT-labeling for differential expression.3,4,6,28 An advantage of applying both ICAT and 2DE approaches to study M. burtonii was the identification of a larger pool of differentially expressed proteins. 21 differentially expressed proteins were identified only in the 2DE study (6 of which did not contain cysteine residues), and 11 were identified only by ICAT analysis. Similar differences between ICAT and 2DE have previously been reported for mammalian cells28 and T. cruzi.6 The use of both 2DE and LC-MS based technologies has also been shown to increase the number of proteins identified from human cells, including those of lower abundance.29,30 Combining technologies is clearly valuable as the application of either 2DE or ICAT is unlikely to exhaustively canvas all differentially expressed proteins. Combining technologies also has merit for interpreting protein expression data that may be affected by posttranslational modification (PTM) of proteins. Numerous examples of PTM in M. burtonii were observed on 2DE gels.20 A good example was for methyl coenzyme M reductase. The R, β, or γ subunits were identified in 8 separate spots that exhibited increased or decreased spot intensities, making it difficult to predict thermal regulation. This was resolved by examining mRNA levels of the three genes, which clearly demonstrated a consistent 3-fold increase at 4 °C. In the present study the ICAT analysis resolved an observation regarding elongation factor 1A (EF-1A) that was present as a spot on 2DE gels only from cells grown at 4 °C. ICAT analysis of EF-1A identified the protein as being equally abundant at both growth temperatures (Table S1). The ICAT analysis is likely to be providing the actual abundance by reporting the level of an unmodified, cysteinecontaining peptide of the protein. In contrast, the apparent differential expression of EF-1A on the 2DE gel is likely to have resulted from the protein being modified. The activity of elongation factor proteins is known to be regulated by GTP

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ICAT proteomics of Methanococcoides burtonii Table 2. Protein Identities and Levels of Differential Expression contig•genea

68•1780 52•360 54•434d 69•1942d 69•1944d 53•401 68•1742 55•482d 70•2399d 69•2017 70•2397* 69•2100 55•472 70•2716d

functional category and gene function

Methanogenesis and energy production Monomethylamine methyltransferase F420 reducing hydrogenase Methylenetetrahydromethanopterin dehydrogenase Trimethylamine methytransferase Trimethylamine corrinoid protein Biosynthesis Glucose-1-phosphate thymidyltransferase Phosphoribosylamine-glycine ligase S-adenosylmethionine synthase Thiamine biosynthesis protein C Transport SufB ABC transporter, ATP-binding protein Information processing TATA-box binding protein Cpn60 Hypothetical Hypothetical: predicted RNA binding protein Hypothetical: CBS and DUF39 domains

no. peptides matched

C12:C13 (4 °C:23 °C)b

MW (kDa)

pI

2 1 1 6 4

22 70 30 36 23

6.0 5.4 5.3 5.3 4.3

2.0 0.4 0.6 0.6 0.6

0.4 0.01 0.15 0.1 0.2

1 1 1 2

44 46 39 46

4.7 4.4 6.4 5.4

0.4 0.5 0.6 0.6

0.08 0.03 0.08 0.15

1

27

6.0

0.3

0.08

1 1

22 54

4.8 4.4

1.7 0.5

0.02 0.15

2 1

5.9 54

8.2 5.1

0.4 0.33

0.1 0.15

SDc

a contig•gene numbers are from the 11Dec03 release of the M. burtonii genome sequence annotation. b Average ratio calculated from 3 independent labeling experiments on 2 separate protein samples. c Standard deviation calculated from relative abundance ratio of the same peptide from the 3 labeling experiments. d Proteins with differential expression levels >1.5-fold in 2 or more labeling experiments

Biological Roles of Differentially Expressed Proteins. The differentially expressed proteins were linked to a range of physiological activities (Table 2). Information about protein abundance at 4 °C, protein identity and gene arrangement were used to make inferences about cold adaptation. In addition, findings from the previous 2DE study20 were compared. Particular attention was paid to the expression levels of individual proteins that were identified using 2DE and ICAT and in particular to those for which the abundance varied by at least 2-fold. The consistency of views that were generated about the regulation of major pathways and processes is highlighted. Energy Generation and Biosynthesis from Methanogenesis. The monomethylamine methyltransferase (MtmB) was at least 2-fold more abundant at 4 °C in all three labeling experiments (Table 2). There is also evidence for upregulation at 4 °C of trimethylamine methyltransferase (MttB) and its cognate corrinoid protein (MttC),20 dimethylamine methyltransferase (MtbB) (Table S1) and increased transcript levels of the dimethylamine corrinoid protein MtbC.20 The previous study also indicated that expression of methylcobalamin:CoM methyltransferase, MtbA (gene 60•801) was enhanced at 4 °C.

Figure 4. Relative abundance of a peptide from Cpn60 identified from M. burtonii grown at 4 °C and 23 °C. (A) Relative abundance of the doubly charged peptide from proteins labeled at 4 °C (L) and 23 °C (H). (B) Tandem mass spectra of a peptide (H) derived from gene 69•2100.

phosphorylation. Consistent with this, trains of spots that vary with pI on 2DE gels have previously been attributed to the phosphorylation state of the identified elongation factor proteins from M. jannaschii.31

Enhanced expression of the methyltransferase machinery at 4 °C implies an increased flux in the direction of methyl coenzyme M production. This key intermediate in methanogenesis participates in a disproportionation reaction, in which the methyl group is either oxidized to CO2 or reduced to CH4. Reducing equivalents from the oxidative reactions are used to reduce the heterodisulfide produced in the reductive reaction, in a process coupled to the generation of a proton motive force. Previous data20 indicated that methyl coenzyme M reductase and coenzyme F420H2 dehydrogenase are upregulated at 4 °C. However, both ATP synthesis and the oxidative branch of methylotrophic methanogenesis appear to be downregulated at 4 °C, as judged by lower amounts of ATP synthase beta subunit (gene 66•1478)20 and methylenetetrahydromethanopterin dehydrogenase (gene 54•434, Table S1)20 at this temperature. Taken together, these results suggest that at lower temperatures, M. burtonii finely tunes the methanogenesis pathway in order to maintain both a proton motive force and Journal of Proteome Research • Vol. 4, No. 2, 2005 477

research articles a minimal level of biosynthetic activity. The complete degradation of trimethylamine to ammonium and the increased flux through methyl coenzyme M to methane reflects these two requirements. The methyltransferases are predicted to be translated from two contiguous open reading frames by the incorporation of pyrrolysine at an in-frame amber stop codon. The genome of M. burtonii contains several copies of the mttB (genes 61•917+61•918 and 69•1942+69•1943), mtbB (genes 51•307+51•308 and 69•1923+69•1924) and mtmB (genes 68•1780+68•1781 and 68•1788+68•1789) genes, encoding tri-, di- and monomethylamine methyltransferases, respectively. The function of pyrrolysine in methylamine methyltransferases has not been experimentally determined. However, for the all three methyltransferases it has recently been proposed to activate and orientate methylamine for methyl transfer to the cobalt ion of the respective corrinoid proteins.32 The effects of temperature on the abundance of the methylamine methyltransferases at 4 °C may reflect a thermodynamic effect on pyrrolysine catalyzed reactions. This may be similar to the inhibitory effect of low temperature on the isomerization of peptide bonds, which appears to be compensated in archaea and bacteria by increasing cellular levels of peptidyl-prolyl cis/ trans isomerases.33,34 Gene 52•360, coenzyme F420-reducing hydrogenase beta subunit, was at least 2-fold more abundant at 23 °C (Table 2). In hydrogenotrophic methanogens, this enzyme oxidizes H2 to generate the reduced electron carrier F420H2. Surprisingly, hydrogenase genes are present in the genomes of methylotrophic methanogens which cannot grow on H2:CO235 and are transcribed under methylotrophic growth conditions.36 The physiological role of the hydrogenase in methylotrophs remains unclear. Upregulation of hydrogenase activity at higher temperatures in M. burtonii suggests either a requirement to generate F420H2 through hydrogen oxidation or perhaps alternatively, a route to hydrogen generation via oxidation of excess reducing equivalents. In this respect, it is noteworthy that hydrogen production has been observed in hydrogenotrophic methanogens under certain growth conditions.37 The levels of a glucose-1-phosphate thymidyltransferase (GPT) were at least 2-fold lower during growth at 4 °C (Table 2). The gene is arranged near a number of other biosynthetic genes (Figure 5). GPT is involved in the addition of nucleotides to sugars. In bacteria it is involved in the synthesis of dTDPL-rhamnose for incorporation in the S-layer.38 Rhamnose is present as a nonlipid sugar in the cell membrane of the methanogen Methanospirillum hungatei,39 and it is therefore possible that GPT is involved in growth temperature specific synthesis of M. burtonii membranes. Consistent with membrane composition being thermally regulated in M. burtonii, lipid unsaturation was recently shown to be growth temperature regulated.17 Phosphoribosylamine-glycine ligase (PGL) levels were found to be reduced during growth at 4 °C (Table 2). Immediately downstream of the gene encoding PGL is a ornithine carbamoyltransferase gene (Figure 5). The increased abundance of PGL may indicate that purine biosynthesis is upregulated at 23 °C. If the relative rates of purine biosynthesis are higher at 23 °C, this may reflect an increased demand for DNA synthesis, consistent with a 4-fold higher growth rate at 23 °C. Thiamine biosynthesis protein was found to be less abundant during growth at 4 °C (Table 2), and a similar finding was made in the previous 2DE study.20 Evidently, biosynthesis and energy 478

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Figure 5. Organization of genes encoding differentially expressed proteins. Expressed proteins (solid arrows) and gene numbers are shown. Ho, hypothetical or conserved hypothetical protein; F420, F420 hydrogenase; MinD, cell division protein; GPT, glucose1-phosphate thymidyltransferase; At, aminotransferase; PGM, phosphoglucomutase; Exo, single stranded DNA exonuclease; UDG, uracil DNA glycosylase; Kin, sugar or amino acid kinase; RNA bp, RNA binding protein; EF1, elongation factor 1; PGL, phosphoribosylamine-glycine ligase; OCT, ornithine carbamoyltransferase; MtmC; monomethylamine corrinoid protein; MtmB, monomethylamine methyltransferase; BioY, biotin synthase; ABC, ABC transporter; Co Tp, cobalt transporter; ArsR, transcriptional regulator; FeS, ferredoxin/[Fe-S] binding protein.

generation pathways (above) are finely and consistently regulated in M. burtonii to accommodate the needs of the cell. To further probe this it may be valuable to examine the expressed proteome in cells growing below 4 °C. We would predict that the cells may reach a stage where metabolism becomes uncoupled from cell division, and methanogenesis is maintained soley for the purpose of fulfilling the cell’s maintenance energy demand. This experimental approach may help to highlight critical features of extreme cold adaptation. Transcription. The TATA-box binding protein (TBP) is fundamentally important for the recruitment of the RNA polymerase complex to the promoter. It is therefore noteworthy that the abundance of this protein was higher at 4 °C. RNA polymerase subunit E and a response regulator member of a two component regulatory system were previously found to be more abundant during growth at 4 °C.20 M. burtonii has two TBPs annotated in the draft genome. It has been suggested that competition between TBPs may provide a mechanism for regulating transcriptional activity, and bacterial-like regulatory proteins may exert their influence through interactions with TBPs and other transcription factors.40 We previously identified all RNA polymerase subunits, and four eucaryotic-like and 18 bacterial-like transcriptional regulatory proteins in the expressed proteome of M. burtonii.20,21 The abundance of transcriptional regulators, and the thermally regulated levels of a

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ICAT proteomics of Methanococcoides burtonii

component of the basal transcription apparatus and two transcriptional regulators, highlights the effect of temperature on the transcription apparatus and the important role that regulation of mRNA levels plays in cold adaptation in M. burtonii. Hypothetical Proteins. The thermal regulation of two hypothetical (Ho) proteins (Table 2) underscores the important role they play in the physiology of the cell, and the general need to increase knowledge of their function.22 Using IPRSCAN, Ho protein 55•472 was predicted to contain a domain that is conserved in archaea and likely to bind zinc through four conserved cysteine residues. Performing BLAST against the COGS database identified matches to COG2888 which contains predicted Zn-ribbon RNA-binding proteins involved in translation. In association with gene organization data showing gene 55•472 immediately upstream of the gene encoding elongation factor 1B (Figure 5), it seems reasonable to suggest that the Ho protein binds RNA and may play a role in protein synthesis. The gene arrangement data is supported by FGENESB analysis which predicts that genes 472 and 473 are likely to form an operon. Ho 70•2716 has a pair of C-terminal CBS domains and a DUF39 domain (of unknown function). The CBS domains thread with high z-scores. Pairs of CBS domains are thought to act as sensors of cellular energy status through the binding of ligands that contain adenosyl moieties, and we previously identified CBS domains in a number of expressed Ho proteins.22 FGENESB predicts gene 70•2716 is in an operon with genes 2717 and 2718, which are annotated as a ferredoxin/[Fe-S] binding protein and a conserved Ho protein, respectively (Figure 5). Using a computational method we recently developed to examine conserved gene context across multiple prokaryotic genomes,22 the 2716/2717 gene pair was found to be conserved in M. jannaschii, Methanosarcina acetivorans, and Methanosarcina mazei. From these analyses it might be speculated that these proteins could function in energy/redox sensing, and they may fulfill a similar role in several methanogens. Stress Indicators During Growth at 23 °C. A SufB homologue was more abundant at 23 °C (Table 2). The sufB gene was arranged in the genome immediately upstream of sufC (Figure 5). sufB and sufC occur in archaea, bacteria and eucaryotes40 and have been reported to be essential genes.42,43 In bacteria, SufB, SufC and SufD form an unorthodox cytoplasmic ABC ATPase that has been implicated in iron acquisition and the assembly of [Fe-S] clusters, particularly in response to oxidative damage.41,44 The elevated levels of SufB in M. burtonii at 23 °C may relate to an increased demand to repair damaged [Fe-S]-containing proteins. M. burtonii encodes three chaperonin, cpn60 genes. All three Cpn60 proteins were detected in the expressed proteome of cells grown at 4 °C.21 In the present study the levels of one of the Cpn60 proteins was found to be at least 2-fold lower at 4 °C (gene 69•2100, Table 2). This cpn60 gene is located adjacent to genes encoding transport proteins, a transcriptional regulator and a hypothetical protein (Figure 5). The other two cpn60 genes are located in different contigs, indicating that the cpn60 genes are not physically arranged to ensure co-regulation of expression. Chaperonins have been shown to be heat shock induced in Pyrococcus furiosus,45, Pyrodictium occultum,46 and Sulfolobus shibatae.47 Moreover, it was recently found in S. shibatae that the expression of two cpn60 genes was increased by heat shock and decreased by cold shock, whereas expression

of a third gene was undetectable at heat shock temperatures but induced by cold shock.48 The authors proposed that in vivo, the oligomeric form of the chaperonin complex was determined by the relative abundance of subunits, highlighting the importance of regulation in directly affecting function. The fact that all three Cpn60 proteins were detected during growth at 4 °C in M. burtonii,21 but the abundance of at least one of the proteins was reduced at 4 °C (Table 2), indicates it is likely that the composition of the chaperonin complex is thermally regulated in M. burtonii. The findings for M. burtonii and S. shibatae indicate that the mechanism of regulating chaperonin complex composition may be conserved in members of the Euryarchaeota (M. burtonii) and Crenarchaeota (S. shibatae) that encode three chaperonin genes. The decreased abundance of the Cpn60 in M. burtonii at 4 °C, and the association of archaeal chaperonins with a heat shock response,49 is consistent with the growth of M. burtonii at 23 °C being a heat stress to the cells. A similar observation was previously made, based on the increased abundance of the DnaK chaperone at 23 °C.20 These observation are consistent with the developing view that elevated temperatures which promote maximum rates of growth (Topt) are stressful for psychrophiles.24,50,51

Conclusion This is the first report of the application of ICAT technology to perform proteomics on any member of the Archaea, and to examine cold adaptation in any organism. The success of the ICAT approach in this organism provides a platform for the application of this technology to both cold-adapted organisms and other members of the methanogenic archaea. Combining different separation, labeling and mass spectrometry technologies20,21 increased the range and number of thermally regulated proteins identified from M. burtonii. This enabled a more comprehensive view of the biology of cold-adaptation in this organism to be developed.

Acknowledgment. We thank Paul Curmi and Haluk Ertan for helpful discussions. The research was supported by the Australian Research Council. Mass spectrometric analysis for the work were carried out at the Bioanalytical Mass Spectrometry Facility, UNSW, and was supported in part by grants from the Australian Government Systemic Infrastructure Initiative and Major National Research Facilities Program (UNSW node of the Australian Proteome Analysis Facility) and by the UNSW Capital Grants Scheme. Supporting Information Available: Supporting Table S1: Proteins Identified by ICAT LC-MS. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (2) Julka, S.; Regnier, F. J. Proteome Res. 2004, 3, 350-363. (3) Guina, T.; Wu, M.; Miller, S. I.; Purvine, S. O.; Yi, E. C.; Eng, J.; Goodlett, D. R.; Aebersold, R.; Ernst, R. K.; Lee, K. A. J. Am. Soc. Mass Spectrom. 2003, 14, 742-751. (4) Shiio, Y.; Donohoe, S.; Yi, E. C.; Goodlett, D. R.; Aebersold, R.; Eisenman, R. N. EMBO J. 2002, 21, 5088-5096. (5) Griffin, T. J.; Han, D. K.; Gygi, S. P.; Rist, B.; Lee, H.; Aebersold, R.; Parker, K. C. J. Am. Soc. Mass Spectrom. 2001, 12,1238-1246. (6) Paba, J.; Santana, J. M.; Teixeira, A. R.; Fontes, W.; Sousa, M. V.; Ricart, C. A. Proteomics 2004, 4, 1052-1059. (7) Ranish, J. A.; Yi, E. C.; Leslie, D. M.; Purvine, S. O.; Goodlett, D. R.; Eng, J.; Aebersold, R. Nat. Genet. 2003, 33, 349-355.

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