Global Proteomic Analysis of the Insoluble, Soluble, and Supernatant

Global Proteomic Analysis of the Insoluble, Soluble, and Supernatant Fractions of the Psychrophilic Archaeon Methanococcoides burtonii Part II: The Effect of Different Methylated Growth Substrates Timothy J. Williams,†,‡ Dominic W. Burg,†,‡ Haluk Ertan,‡,§ Mark J. Raftery,| Anne Poljak,|,⊥ Michael Guilhaus,| and Ricardo Cavicchioli*,‡ School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, 2052, NSW, Australia, Department of Molecular Biology and Genetics, Science Faculty, Istanbul University, Vezneciler, Istanbul, Turkey, Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Sydney, 2052, NSW, Australia, and School of Medical Sciences, The University of New South Wales, Sydney, 2052, Australia Received June 10, 2009

Methanococcoides burtonii is a cold-adapted methanogenic archaeon from Ace Lake in Antarctica. Methanol and methylamines are the only substrates it can use for carbon and energy. We carried out quantitative proteomics using iTRAQ of M. burtonii cells grown on different substrates (methanol in defined media or trimethylamine in complex media), using techniques that enriched for secreted and membrane proteins in addition to cytoplasmic proteins. By integrating proteomic data with the complete, manually annotated genome sequence of M. burtonii, we were able to gain new insight into methylotrophic metabolism and the effects of methanol on the cell. Metabolic processing of methanol and methylamines is initiated by methyltransferases specific for each substrate, with multiple paralogs for each of the methyltransferases (similar to other members of the Methanosarcinaceae). In M. burtonii, most methyltransferases appear to have distinct roles in the metabolism of methylated substrates, although two methylamine methyltransferases appear to be nonfunctional. One set of methyltransferases for trimethylamine catabolism appears to be membrane associated, potentially providing a mechanism to directly couple trimethylamine uptake to demethylation. Important roles were highlighted for citrate synthase, glutamine synthetase, acetyl-CoA decarbonylase/synthase, and pyruvate synthase in carbon and nitrogen metabolism during growth on methanol. M. burtonii had only a marginal response to the provision of exogenous amino acids (from yeast extract), indicating that it is predisposed to the endogenous synthesis of amino acids. Growth on methanol appeared to cause oxidative stress in the cell, possibly through the formation of reactive nonoxygen species and formaldehyde, and the oxidative inactivation of corrinoid proteins, with the cell responding by elevating the synthesis of universal stress (Usp) proteins, several nucleic acid binding proteins, and a serpin. In addition, changes in levels of cell envelope proteins were linked to counteracting the disruptive solvent effects of methanol on cell membranes. This is the first global proteomic study to examine the effects of different carbon sources on the growth of an obligately methylotrophic methanogen. Keywords: proteome • LC/LC-MS/MS • iTRAQ • archaea • methanogen • psychrophile • methylotroph • methylamine • methanol • methyltransferase • cell envelope • nucleic acid binding • oxidative stress

Introduction Methane-producing archaea (methanogens) play a unique role in the global carbon cycle, generating the C1 compound methane as the final reduced form of carbon in anaerobic * To whom correpondence should be addressed. Rick Cavicchioli, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, 2052, NSW, Australia. E-mail [email protected]; Tel. (+61) 2 9385 3516; Fax (+61) 2 9385 2742. † These authors contributed equally. ‡ School of Biotechnology and Biomolecular Sciences, The University of New South Wales. § Istanbul University. | Bioanalytical Mass Spectrometry Facility, The University of New South Wales. ⊥ School of Medical Sciences, The University of New South Wales. 10.1021/pr9005102

 2010 American Chemical Society

environments.1 By producing methane as a byproduct of their energy generation pathways, methanogens in cold environments (e.g., sediments, permafrost) are capable of making significant contributions to global carbon emissions.2 Understanding how these methanogens respond to changes in temperature is important for being able to forecast the contribution that methane will make to global carbon levels. Improving the understanding of how temperature regulates methanogenesis will also provide opportunities for determining effective ways of harnessing methane as an energy source from the growth of methanogens in cold environments. Methanococcoides burtonii is a methanogen that was isolated from cold (1-2 °C), anaerobic waters in Ace Lake, Antarctica.3 M. burtonii has proven to be a useful model for studying cold Journal of Proteome Research 2010, 9, 653–663 653 Published on Web 12/01/2009

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adaptation in archaea, with proteomic and genomic analyses providing valuable insights into cold adaptive mechanisms. To better determine the extent and abundance of proteins globally synthesized in M. burtonii, proteomics has been expanded to include the analysis of insoluble fractions, in addition to soluble and supernatant fractions, providing novel avenues for examining the adaptive responses of the organism (Part I11). In natural environments microorganisms respond to a range of biotic and abiotic factors. Nutrient limitation and substrate utilization are key factors controlling growth, competition, and community composition. Both methanol and trimethylamine (TMA) are potential substrates for methanogens in marine sediments,12 although both compounds are likely to be of relatively low abundance.13 Although the free energy (∆G) yield per mole of methane is higher for growth on methanol than TMA,14 TMA has higher free energy per mole of substrate, and is preferred over methanol for growth.15 TMA also has the advantage of serving as both a carbon and nitrogen source, whereas growth on methanol must be accompanied by a nitrogen source, such as ammonia.10 The metabolism of M. burtonii remains relatively poorly understood, with all studies to date focusing on growth with TMA as the carbon source. In contrast to the metabolic versatility exhibited by Methanosarcina spp.,16-18 and to the majority of methanogens, which are capable of autotrophic growth,1 M. burtonii is limited to growth on methanol and methylamines; it cannot grow on hydrogen, CO2, formate, acetate, or methylthiols.3,10 Previous studies on Methanosarcina thermophila and Methanosarcina acetivorans have identified quantitative changes in protein abundances between cells grown on acetate and methanol.19-22 Many of the proteins identified are involved in the respective pathways for acetate and methanol utilization. However, in one study, proteins not directly involved in metabolism were also identified, including the increased abundance of chaperone proteins for M. acetivorans grown on methanol.22 Methanol has two potentially damaging effects on cells. First, as a solvent, it can disrupt cell membranes and thereby threaten the structural and functional integrity of the cell.23,24 For M. burtonii, these effects would be expected to be manifested at the cell envelope. In M. burtonii, and many other archaea, the cell envelope includes a proteinaceous surface layer (S-layer) that is anchored to the cytoplasmic membrane and separated from it by a “quasi-periplasmic space”.25 Second, the enzymatic oxidation of methanol (e.g., by alcohol dehydrogenase) can also generate the highly reactive compounds formaldehyde and peroxide, causing damage to lipids, DNA and other intracellular macromolecules.26 Under the strict anaerobic conditions in which M. burtonii grows, reactive oxygen species cannot be generated. However, the synthesis of oxidative stress proteins to combat other toxic compounds or reactive nonoxygen species may conceivably be produced as a consequence of anaerobic metabolism. The proteomic methods employed to enrich for integral membrane, secreted and cell surface proteins for cells growing on methanol or TMA (see Part I11) provided the means for evaluating the global cellular response to these methylated substrates. This is the first proteomic study to examine the effects of carbon sources on the growth of an obligately methylotrophic methanogen. 654

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Experimental Procedures M. burtonii (DSM 6242) cultures were grown anaerobically in a complex medium containing TMA (MFM) or a defined medium containing methanol (M-medium) at 4 or 23 °C. Cultures were harvested and three fractions prepared: supernatant (enriched for secreted proteins), insoluble (enriched for integral membrane proteins, membrane-associated proteins, and dense macromolecular complexes), and soluble. Proteins were labeled using the 4plex iTRAQ postincorporation labeling system.27 Peptides were analyzed by LC/LC-MS/MS using an API QStar Pulsar i hybrid tandem mass spectrometer. MS data were searched using Mascot and data assessed using a false discovery rate, and iTRAQ abundance data processed using ProQuant software. All protein identifications were linked to manually annotated gene assignments. A complete description of the experimental procedures is described in Part I.11

Results and Discussion 1. Methanogenesis, Central Metabolism, and Energy Conservation. M. burtonii obtains energy and biosynthetic carbon by converting the methyl groups of methylamines and methanol to methane and CO2.3 Methylamines are generated from the degradation of algae and bacteria (especially osmolytes28), and methanol can be generated as a byproduct of aerobic methanotrophy, such as occurs in Ace Lake.29 The previous identification of MttP as a TMA permease30 is consistent with our proteomic data, but there is no identifiable mechanism for methanol uptake. Similar to methylotrophic bacteria, it appears that TMA uptake is mediated by a specific transporter in M. burtonii, whereas methanol presumably enters the cell by diffusion.31 Both MttP permeases were detected in M. burtonii, and the levels of one (MttP-1) were elevated for cells grown on TMA vs methanol (Table 1). The disproportionation of methanol is initiated by the transfer of the methyl group to the corrinoid cofactor bound to a specific methanol methyltransferase (methanol-MT), MtaBC (including corrinoid protein), with subsequent transfer to coenzyme M (CoM) by methanol:CoM MT (MtaA) (Figure 1).32 The catabolism of TMA involves the sequential conversion of TMA to dimethylamine (DMA) and monomethylamine (MMA), with the successive demethylations catalyzed by MTs (including corrinoid proteins) that are specific for each substrate: MttBC (TMA-MT), MtbBC (DMA-MT), and MtmBC (MMA-MT).30,33-35 A single enzyme (MtbA, methylamine:CoM MT) catalyzes the methyl transfer from each of these corrinoid proteins to CoM to generate methyl-CoM, the key intermediate in both the methanol and methylamine methyl transfer pathways.32 The methyl moiety of methyl-CoM can either be directly liberated as methane by methyl-CoM reductase (Mcr) and coenzyme B, or transferred to tetrahydrosarcinapterin (H4SPT) by a specific MT complex (Mtr).17 For every methyl group that is oxidized to CO2 by the latter pathway, six electrons are generated that can be used to reduce three other methyl groups to methane. The electron acceptors in the oxidative branch of this disproportionation reaction are F420 and ferredoxin; the membranebound F420H2 dehydrogenase (Fpo) generates a proton gradient that drives ATP synthesis.36 Carbon assimilation for biosynthesis proceeds via the acetyl-CoA decarbonylase/synthase (ACDS) complex, which generates acetyl-CoA from the condensation of the methyl moiety of methyl-H4SPT and endogenous CO2 (Figure 1). The abundance of proteins identified in the expressed proteome clearly reflected the substrate-utilization pathways

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Part II: The Effect of Different Methylated Growth Substrates

Table 1. Differentially Abundant Methanogenesis Proteins in the Expressed Proteome of M. burtonii During Growth at 23 versus 4 °C Differential abundance (TMA/Methanol) 4 °C locus tag

protein functiona

sizeb

TMDc

soluble

23 °C

insoluble

S/natant

soluble

insoluble

S/natant

3.7 ns ns ns 2.3 3.2 ns ns ns ns ? ? ns ns ns

5.4 5.0 2.2 3.3 ns 3.6 ns ns ? ? 1.8 2.7 ns

4.2 7.6 3.5 3.4 ns ns 4.8 12 2.7 ? ? 2.1 1.6 ns

3.3 ns ns ns ns ns 5.8 ns ns ? ? ns ns ns

2.5 4.7 2.8 3.0 ns 4.4 8.7 ns ns ? ? 2.0 ns ns

ns ns

1.8 -

1.6 ns

ns ns

1.5 -

ns 0.24 ns -

0.24 0.13 0.24 ns

0.06 0.06 0.11 0.25

0.18 0.10 ns -

0.15 0.11 0.16 0.20

0.65

ns

ns

1.5

ns

1.8 ns

ns -

1.9 ns

2.9 1.5

ns -

Methanogenesis Mbur_1369 Mbur_1368 Mbur_1364 Mbur_1365 Mbur_1367 Mbur_1366 Mbur_2308 Mbur_2310 Mbur_2313 Mbur_2311 Mbur_2312 Mbur_2291 Mbur_2288 Mbur_0839 Mbur_0846 Mbur_0840/ Mbur_0847 Mbur_2082 Mbur_1370 Mbur_0814 Mbur_0813 Mbur_0812 Mbur_0811 Mbur_0808 Mbur_0809 Mbur_2417 Mbur_2420 Mbur_2437 Mbur_0929 Mbur_1518 Mbur_1525

Methylamine-specific 496 0 3.8 TMA-MTe, MttB (ER2) (MttB-1) TMA CPf, MttC (ER2) (MttC-1) 216 0 11 DMA-MT, MtbB (ER2) (MtbB-1) 467 0 2.6 DMA CP, MtbC (ER2) (MtbC-1) 251 0 3.3 TMA permease, MttP (ER4) (MttP-1) 348 9 ns MttQ (function unknown) (ER4) (MttQ-1) 98 0 ns TMA-MT, MttB (ER2) (MttB-2) 497 0 3.3 TMA CP, MttC (ER2) (MttC-2) 216 0 ns TMA-MT, MttB (ER2) (MttB-3) 484 0 TMA permease MttP (ER4) (MttP-2) 347 9 ns MttQ (function unknown) (ER4) (MttQ-2) 98 0 ns DMA-MT, MtbB (ER2) (MtbB-2) 187 0 ? DMA CP, MtbC (ER2) (MtbC-2) 168 0 ? MMA-MT, MtmB (ER2) (MtmB-1) 458 0 2.5 MMA-MT, MtmB (ER2) (MtmB-2) 458 0 ns MMA CP, MtmC (ER2) (MtmC-1 or MtmC-2) 217 0 1.7 Methylamine:CoM MT, MtbA (ER2) RamA (ER2)

344 540

0 0

ns 0.58

Methanol-specific Methanol-MT, MtaB (ER2) (MtaB-1) 461 0 0.16 Methanol CP, MtaC (ER2) (MtaC-1) 256 0 0.12 Methanol MT, MtaB (ER2) (MtaB-2) 462 0 Methanol CP, MtaC (ER2) (MtaC-2) 254 0 Methanol:CoM MT, MtaA (ER2) 338 0 0.21 RamM (ER2) 535 0 0.31 Common methanogenesis proteins Methyl-CoM reductase, R subunit, 572 0 ns McrA (ER2) McrD (function unknown) (ER4) 162 0 ns CoB--CoM heterodisulfide reductase, 415 0 1.6 subunit D, HdrD (ER2) Methylene-H4SPTg dehydrogenase 273 0 ns (Mtd) (ER2) H4SPT S-MT subunit H, MtrH (ER2) 318 0 ns H4SPT S-MT subunit E, MtrE (ER2) 301 5 ns

ns

1.9

ns

ns

ns

ns ns

ns 0.58

ns -

2.2 0.52

ns -

ns ns ns ns

ns 0.33 ns ns

0.65 ns 0.60 0.61

ns ns ns ns

ns 0.63 0.43 ns

ns 0.29 0.06 0.54 ns

ns ns ns 0.56 0.65 0.56

ns ns ns -

ns 0.55 ns 0.56 ns

ns

0.64

0.63 ns

ns 0.55

ns ns

0.56 ns

ns 0.60 ns

0.56 ns 0.54

ns ns

ns ns ns

General metabolism Mbur_2155 Mbur_2157 Mbur_0860 Mbur_1075

Pyruvate synthase, γ subunit (ER2) Pyruvate synthase, R subunit (ER2) ACDS, β subunit (ER2) Re-Citrate Synthase (ER2)

Central carbon metabolism 180 0 ns 404 0 ns 470 0 0.44 425 0 0.62

Mbur_0514

Ammonia assimilation and amino acid synthesis Glutamate dehydrogenase (ER2) 416 0 1.7 ns Glutamine synthetase (ER2) 442 0 ns LL-diaminopimelate aminotransferase (ER2) 385 0 ns Ketol-acid reductoisomerase (ER2) 335 0 ns ns D-3-phosphoglycerate dehydrogenase (ER2) 523 0 ns ns 2-amino-3,7-dideoxy-D-threo-hept-6-ulosonate 263 0 0.63 synthase (ER3) Aminotransferase, class V (AGAT family) (ER3) 380 0 0.58 ns

Mbur_1969 Mbur_1994

Gluconeogenesis and ribulose monophosphate pathway Fructose-bisphosphate aldolase, class II (ER3) 379 0 ns ns Fae/hps bifunctional enzyme (ER2) 396 0 ns ns

Mbur_1330 Mbur_1494 Mbur_0421

SAM synthetase (ER2) ThiC (ER2) PLP synthase, lyase subunit (ER2)

Mbur_1973 Mbur_1975 Mbur_1013 Mbur_0708 Mbur_2385 Mbur_0917

Cofactor biosynthesis 401 0 427 0 298 0

0.55 0.59 ns

ns ns

ns

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Table 1. Continued Differential abundance (TMA/Methanol) 4 °C locus tag

protein functiona

sizeb

TMDc

soluble

insoluble

23 °C S/natant

131 Other

0

2.1

-

ns

455

0

1.5

-

ns

1.5 ns

ns

-

ns 4.7 ns 1.9 ns -

soluble

insoluble

S/natant

-

ns

ns

-

ns

ns ns

ns 0.36

ns

ns ns

0.55 -

ns

-

ns 0.53

ns

ns ns 1.7 ns 0.63 0.47

ns ns ns ns ns -

ns ns ns ns 1.6 -

2.9 ns ns ns 1.6

ns ns ns ns ns -

368 0 0.67 Unknown (DNA- or RNA-binding)

2.2

ns

ns

ns

ns

68 0 67 0 63 0 500 0 79 0 426 0 475 0 243 0 447 0 Stress proteins

0.30 0.5 0.49 ns 0.46 ns 0.44 ns ns

ns ns ns ns 1.7 ns ns

0.46 0.64 0.38 ns 0.22 ns 0.42 ns

ns 0.43 0.45 ns ns 0.58 ns 0.66 0.68

ns ns ns ns 1.6 ns ns

0.66 0.58 0.47 0.56 ns ns ns ns

Universal stress protein UspA/UspF (ER3) Universal stress protein UspA (ER4) Peroxiredoxin (ER2) Rubrerythrin domain protein (ER) Secreted and

150 0 ns 176 0 ns 228 0 4.6 189 0 0.57 cell surface proteins

ns -

ns -

0.64 0.57 0.6 ns

ns -

0.49 -

Cadherin domain protein (ER4) Ig-like domain protein (ER4) YVTN/NHL protein (ER3) YVTN/NHL protein (ER3) PepSY-like domain protein (ER4) Trypsin-like serine protease (ER4) MxaI/MoxI-like protein (ER4) Protein with duplicated DUF1608 (ER4) Protein with duplicated DUF1608 (ER4) Serpin (serine proteinase inhibitor) (ER3) Serpin (serine proteinase inhibitor) (ER3) SPFH domain protein (ER4) SPFH domain protein (ER3) Flagellin (ER2) Flagellin (ER2)

1021 462 468 415 401 368 167 867 677 450 420 386 252 259 190

ns ns 0.02 0.15 2.8 ns ns ns ns

ns 0.29 ns ns ns 0.6 ns ns ns 0.63 ns 0.51 -

ns 0.69 0.47 0.54 0.47 1.8 0.53 ns 0.51 ns ns

ns ns ns 0.04 0.05 ns ns ns ns

ns 0.34 ns 0.24 0.16 0.16 0.56 0.49 0.17 0.53 0.61 0.41 -

0.4 0.22 ns ns 0.32 0.52 0.22 ns 0.34 ns 0.19

Mbur_1035

Sirohydrochlorin cobaltochelatase, CbiXS (ER2)

Mbur_1328 Mbur_0586 Mbur_1787

Sulfide dehydrogenase (flavoprotein) subunit SudA (ER2) FMN-binding protein (ER4) FeS cluster assembly protein (ER4)

Mbur_0994 Mbur_1363

PPi-energized proton pump, HppA (ER2) Sodium/hydrogen antiporter (ER4)

Mbur_0963 Mbur_0039 Mbur_1177 Mbur_2100 Mbur_0199 Mbur_0245 Mbur_1950

TFB (ER2) 337 RNA polymerase subunit D, RpoD (ER2) 267 RNA polymerase subunit A′, RpoA1 (ER2) 883 Transcriptional regulator, AsnC/Lrp family (ER3) 163 Exosome RNA-binding protein, Rp41 (ER2) 343 DEAD-box RNA helicase (ER2) 463 DEAD box RNA helicase (ER2) 522 Replication

Mbur_1942

Cell division protein, FtsZ (ER2)

Mbur_0304 Mbur_0604 Mbur_1445 Mbur_0311 Mbur_1512 Mbur_1362 Mbur_1769 Mbur_2095 Mbur_2398

TRAM domain protein (ER4) TRAM domain protein (ER4) TRAM domain protein (ER4) CBS and DUF39 protein (ER4) Winged helix protein (ER4) SSB protein (ER3) Toprim domain protein (ER4) aRadC (ER4) RNase J-like protein ER4)

Mbur_2350 Mbur_2183 Mbur_0959 Mbur_2382 Mbur_0314 Mbur_2003 Mbur_0060 Mbur_1111 Mbur_1112 Mbur_1349 Mbur_0714 Mbur_0268 Mbur_1690 Mbur_1742 Mbur_0702 Mbur_1185 Mbur_1804 Mbur_0104 Mbur_0346

134 0 107 0 Energy conservation 672 16 610 13 Information processing

1.6

Transcription 0 0 0 0 0 0 0

1 1 1 0 0 0 1 1 1 0 0 1 1 1 1

a Protein function based on manual annotations and assigned an Evidence Rating value (Allen et al. 2009). b Size in number of amino acids. c TMD, number of predicted transmembrane domains. d Differential abundance values are expressed as ratios, for the two temperatures (23 °C/4 °C) for each growth medium, for three fractions (soluble, insoluble, supernatant [S/natant]). NS, detected in the proteome but no significant difference. -, not detected in the expressed proteome. e All secreted and cell surface proteins have a signal peptide sequence identified by SignalP. f CP, corrinoid protein. g H4SPT, tetrahydrosarcinapterin.

specific to the methylated carbon source provided in the growth medium, while proteins common to methanol and TMA metabolism were generally detected with equivalent abundance (Table 1; Figure 1). Further, the abundance of enzymes specific to methanol and TMA methyl transfer reactions tended to increase at 23 °C, which is likely to reflect the higher rates of substrate specific metabolism required to maintain higher rates of growth (Table 2). A number of proteins involved in transcription were more abundant in TMA-grown cells, including transcription factor B, two RNA polymerase subunits, a tran656


scriptional regulator (AsnC/Lrp family), and a component of the exosome complex that is involved in mRNA turnover. This is consistent with an increased demand for gene expression at higher rates of growth, including high level expression of a larger number of genes required for the processing of TMA compared to methanol (Figure 2). 1.1. Paralogous Methyltransferases. The M. burtonii genome encodes multiple paralogs of substrate-specific MT genes: methanol-MT (two MtaB and MtaC paralogs), TMA-MT (three MttB and two MttC paralogs), DMA-MT (three MtbB and


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Figure 1. Methanogenesis pathways in M. burtonii. Differentially abundant proteins in Methanococcoides burtonii according to growth medium; proteins higher in methanol-grown cells are shown in green, proteins higher in trimethylamine-grown cells are shown in purple. Protein that were detected but were not differentially abundant are shown in blue. Green dotted box encloses the methanolspecific steps; purple dotted box encloses the methylamine-specific steps. Stippled arrows indicate passive diffusion across a membrane. Abbreviations: ACDS, acetyl-CoA decarbonylase/synthase; Fd, ferredoxin; Fmd (Mo), formylmethanofuran dehydrogenase (molybdenum); Fpo, F420H2 dehydrogenase; Ftr, formylmethanofuran-H4SPT formyltransferase; GDH, glutamate dehydrogenase; GS, glutamine synthetase; H4SPT, tetrahydrosarcinapterin; Hdr, CoB-CoM heterodisulfide reductase; Mch, methenyl-H4SPT cyclohydrolase; Mcr, methylcoenzyme M reductase; Mer, coenzyme F420-dependent methylene-H4SPT reductase; MFR, methanofuran; MP, methanophenazine; MtaA, methanol:CoM methyltransferase; MtaB, methanol methyltransferase; MtaC, methanol corrinoid protein; MtbA, methylamine:CoM methyltransferase; MtbB, dimethylamine methyltransferase; MtbC, dimethylamine corrinoid protein; Mtd, methylene-H4SPT dehydrogenase; MtmB, monomethylamine methyltransferase; MtmC, monomethylamine corrinoid protein; Mtr, H4SPT S-methyltransferase; MttB, trimethylamine methyltransferase; MttC, trimethylamine corrinoid protein; MttP, trimethylamine permease; RamA, methylamine: CoM methyl transfer reductive activation protein; RamM, methanol/CoM methyl transfer reductive activation protein.

MtbC paralogs), and MMA-MT (two MtmB and MtmC paralogs) (Table 3). Only one of the MtaB-MtaC pairs (MtaB-1, MtaC-1) was detected in the proteome, despite all of the methanolspecific MT genes being arranged in a single gene cluster (Figure 2). The genes for the TMA-, DMA-, and MMA-MT enzymes are located in three discrete gene clusters, two of which contain TMA- and DMA-MT genes and a third which contains the MMA-MT genes (MtmBC). Two TMA-MTs (MttB-1, MttB-2) were detected in the proteome, along with their cognate corrinoid proteins (MttC-1, MttC-2), but the third TMA-MT, MttB-3, was not. In methanogenic archaea an amber codon

within the MT genes encodes a pyrrolysine residue, with the pyrrolysine playing an essential role in methyl transfer reactions involving methylamine substrates.37 The genes for pyrrolysine synthesis and pyrrolysyl-tRNA synthetase are located within a cluster that contains the gene for MtbA, and all the protein products of these genes were identified in the proteome, although only MtbA was differentially abundant (Figure 2). Among the seven TMA-, DMA-, or MMA-MT genes, mttB3 (Mbur_2313) is the only one that lacks an internal amber (TAG) codon. The absence of MttB-3 in the expressed proteome is consistent with MttB-3 not being synthesized with pyrrolysine and not being able to function in TMA metabolism. Journal of Proteome Research • Vol. 9, No. 2, 2010 657

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Table 2. Differentially Abundant Proteins in the Expressed Proteome of M. burtonii During Growth in Medium Containing TMA (MFM) or Methanol (M-medium) Differential abundance (23 °C/4 °C)b TMA locus tag

protein function

a

soluble

Methanol

insoluble

S/natant

soluble

insoluble

S/natant

0.49 0.52 NS 0.63 NS 2.8 2.7 1.6 NS NS

2.4 NS NS NS 2.8 2.2 NS 1.8 NS 3.3

0.65 NS NS NS 2.2 2.2 NS 1.9 NS

0.64 NS 0.63 0.62 NS NS NS NS NS NS

6.9 5.3 NS NS NS NS NS 0.35 NS NS

0.53 NS 0.41 NS NS NS NS 1.6 1.6

MMA CP, MtmC (ER2) (MtmC-1 or MtmC-2)

NS

NS

1.6

NS

NS

NS

Methylamine:CoM MT, MtbA (ER2)

NS

2.6

1.7

NS

2.5

NS

NS 1.7 NS

NS NS NS

0.14 0.06 NS

2.1 2.0 NS

5.6 3.3 NS

2.8 3.0 1.8

2.2 2.6 0.61 2.4 2.0 2.6 2.6 NS

NS NS 1.6 NS

NS NS NS NS NS 0.64

NS 2.2 5.2 3.4 NS NS NS NS

NS 1.6 NS 0.58

Methylamine-specific Mbur_1369 Mbur_1368 Mbur_1364 Mbur_1365 Mbur_1367 Mbur_2308 Mbur_2310 Mbur_2312 Mbur_0839 Mbur_0846 Mbur_0840/ Mbur_0847 Mbur_2082

TMA-MT, MttB (ER2) (MttB-1) TMA CP, MttC (ER2) (MttC-1) DMA-MT, MtbB (ER2) (MtbB-1) DMA CP, MtbC (ER2) (MtbC-1) TMA permease, MttP (ER4) (MttP-1) TMA-MT, MttB (ER2) (MttB-2) TMA CP, MttC (ER2) (MttC-2) MttQ (function unknown) (ER4) (MttQ-2) MMA-MT, MtmB (ER2) (MtmB-1) MMA-MT, MtmB (ER2) (MtmB-2)

Methanol-specific Mbur_0814 Mbur_0813 Mbur_0808

Methanol-MT, MtaB (ER2) (MtaB-1) Methanol CP, MtaC (ER2) (MtaC-1) Methanol:CoM MT, MtaA (ER2)

Mbur_2417 Mbur_2418 Mbur_2420 Mbur_2421 Mbur_2437 Mbur_1288 Mbur_1291 Mbur_2371

Common methanogenesis proteins Methyl-CoM reductase, R subunit, McrA (ER2) NS Methyl-CoM reductase, γ subunit, McrG (ER2) NS McrD (function unknown) (ER4) NS Methyl-CoM reductase, β subunit, McrB (ER2) NS CoB-CoM heterodisulfide reductase, subunit D, HdrD (ER2) NS F420H2 dehydrogenase subunit M, FpoM (ER2) F420H2 dehydrogenase subunit J, FpoJ (ER2) F420H2 dehydrogenase subunit F, FpoF (ER2) NS

a Protein function based on manual annotations and assigned an Evidence Rating value (Allen et al. 2009). b Differential abundance values are expressed as ratios, for the two temperatures (23 °C/4 °C) for each growth medium, for three fractions (soluble, insoluble, supernatant [S/natant]). NS, detected in the proteome but no significant difference. -, not detected in the expressed proteome. All other abbreviations as for Table 1.

The DMA-MT gene mtbB2 (Mbur_2291) is interrupted by a transposon that also introduces a stop codon (TAA) soon after the insertion site and is not likely to produce a functional protein product. The protein sequences of MtbB-2 (if expressed) and the full-length MtbB-1 are almost identical up to the insertion site in the former, which prevented unambiguous peptide discrimination of the two proteins (Table 3). However, the detection of peptides matching the C-terminal region of the protein confirms that MtbB-1 is synthesized, as this region encoded in mtbB2 follows the inserted transposon. Peptide matches to the corrinoid proteins for DMA-MT and MMA-MT were obtained, but again due to their similarity, the two genes for each MT were not able to be distinguished from one another (Table 3). In contrast to DMA-MTs, unique peptide signatures for each of the MMA-MTs (MtmB-1, MtmB-2) were detected, confirming both genes were expressed. Thus, M. burtonii TMA demethylation appears to utilize two TMA-MT paralogs, only one DMA-MT paralog, and two MMA-MT paralogs. For the M. burtonii TMA-MTs, MttB-1 was more abundant in the insoluble fraction and less abundant in the soluble fraction at 23 vs 4 °C in both growth media. MttC-1 showed a similar abundance profile, as did the DMA-MT paralog MtbB1, which were both encoded by the same gene cluster as mttB1. However, MttB-2 and MttC-2 were more abundant across all fractions in cells grown on TMA at 23C vs 4 °C. This may indicate that during growth at 23 °C, MttB-1 preferentially associates with the cell membrane, whereas the cellular location of MttB-2 does not change appreciably. Furthermore, the 658


TMA permease MttP-1 was also more abundant in the insoluble fraction at 23 °C, whereas MttP-2 showed no response to temperature, or to substrate. These data are consistent with one MttBC isozyme (MttBC-1) preferentially associating with the TMA permease, MttP-1, in the membrane during growth at 23 °C. This might be a mechanism to improve the efficiency of TMA catabolism by directly coupling uptake with demethylation. Regulation of this process would be facilitated by the arrangement of the genes in an operonlike gene cluster (Figure 2). Adjacent to each of the two mttP TMA permease genes are short (98 amino acids) open reading frames that are highly conserved in terms of sequence identity and gene arrangement in the Methanosarcinales. The sequence of both proteins is very similar (93%; Table 3) and the only identifiable feature is a possible signal peptide, which may indicate that they are attached to the inner surface of the cell membrane. Both proteins were detected in the proteome, providing evidence for the first time that they are expressed and likely to be functional: here they have been designated as MttQ-1 and MttQ-2 (matching the numbering for the respective MttP permeases encoded by the adjacent gene; Figure 2). Both were more abundant in TMA-grown cells: MttQ-1 in the insoluble fraction and MttQ-2 in the soluble fraction, mirroring the abundance pattern of their associated MttP permeases. It therefore seems likely that MttQ associates with the permease and other cognate members of the MT-I complex and plays a functional role in methyl transfer


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Figure 2. Arrangement of genes associated with the methylamine- or methanol-specific pathways of methanogenesis in M. burtonii. a Locus tags are shown above the genes, protein products below. Genes for which the protein products were differentially abundant according to growth medium are shown in purple (higher in TMA-grown cells) or green (higher in methanol-grown cells). Genes for which the protein products were detected but not differentially abundant are shown in blue. It is not known if MtbB-2/Tn and MtbC-2 are expressed. Proteins that are inferred to be nonfunctional are indicated with a red X.

reactions, perhaps in mediating interactions between the TMA permease and MT products encoded by their respective gene clusters. Multiple copies of MT genes have been identified in other methanogenic archaea.18,20,21,29,38 It has been proposed that the multiple copies may be remnants of past duplication events and provide no selective advantage to the species harboring them.20 In M. burtonii, the disruption of MtbB-2 by a transposon, and the lack of a site for pyrrolysine incorporation in MttB-3, rendering both nonfunctional as MTs, is consistent with this suggestion. However, there is experimental evidence that MT isozymes facilitate efficient switching between sub-

strates,19-21,39 including methanol to TMA,15 and the response to other growth conditions (e.g., substrate concentration).15,39-41 This suggests that isozymes have distinct roles in the metabolism of the methylated substrates that they are specific for. The proteomic data for M. burtonii indicate that differences in the metabolic function of methylamine MT isozymes may extend to, and be facilitated by, their subcellular localization. Overall, our analyses indicate that two MT paralogs of M. burtonii represent nonfunctional remnants, while the majority fulfill roles in the fine-tuning of a methylotrophic metabolism. 1.2. Formylmethanofuran Dehydrogenase Complex. The terminal step in the M. burtonii oxidative branch of the Journal of Proteome Research • Vol. 9, No. 2, 2010 659

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Williams et al.

Table 3. Sequence Identities of Paragalous Methanogenesis Proteins in M. burtonii namea

paralogs

sequence identity (%)

TMA-MT TMA-MT TMA-MT TMA CP DMA-MT DMA CP MMA-MT MMA CP Methanol-MT Methanol CP TMA permease s

MttB-1 vs MttB-2 MttB-1 vs MttB-3 MttB-2 vs MttB-3 MttC-1 vs MttC-2 MtbB-1 vs MtbB-2b MtbC-1 vs MtbC-2 MtmB-1 vs MtmB-2 MtmC-1 vs MtmC-2 MtaB-1 vs MtaB-2 MtaC-1 vs MtaC-2 MttP-1 vs MttP-2 MttQ-1 vs MttQ-2

68 26 28 98 97 94 75 99 70 60 78 93

a Abbreviations as for Table 1. b Paralog disrupted by transposon insertion; sequence identity determined over 187 amino acids of the aligned sequence.

methylotrophic pathway is catalyzed by the formylmethanofuran dehydrogenase complex (FD), which yields CO2 from formylmethanofuran. The genome encodes two FD complexes, organized as two operon-like clusters: FmdEFACBD, for the molybdenum-dependent FD (Fmd), arranged in a similar way to M. barkeri (fmdEFACBD42) and a tungsten-dependent FD (Fwd), arranged in a similar way to Methanopyrus kandleri (fwdGBD43), although containing the cysteine (fwcB), rather than selenocysteine (fwuB) subunit. Only components of Fmd were detected, and protein abundance was not influenced by growth conditions. In Methanobacterium thermoautotrophicum the Fwd genes are constitutively expressed.44 It remains to be determined whether adding tungsten to the growth media would lead to detectable Fwd proteins in M. burtonii. 1.3. General Carbon and Nitrogen Metabolism. Growth on methanol led to higher citrate synthase levels (Table 1). M. burtonii has an incomplete oxidative tricarboxylic acid cycle terminating at 2-oxoglutarate.10 Citrate synthase catalyzes the rate-limiting step of this pathway, and may be key in regulating the supply of 2-oxoglutarate. Increased 2-oxoglutarate synthesis signals perceived nitrogen deficiency and stimulate GS activity, and may thereby promote the assimilation of scarce ammonium.45 The M. burtonii genome encodes genes for ammonia assimilation via the glutamine synthetase/glutamate synthase pathway (GS-GOGAT), or glutamate dehydrogenase (GDH).10 Growth on TMA was accompanied by a higher abundance of GDH, whereas growth on methanol led to a higher abundance of GS (Table 1). Metabolization of TMA liberates ammonia into the cytoplasm, whereas growth on methanol requires the uptake of ammonia. The ammonia transporter (Amt-3; Mbur_0933) in M. burtonii is predicted to be functionally impaired and exogenous ammonia is expected to be imported by diffusion,10 which may restrict the levels of available intracellular ammonia and therefore favor assimilation by high-affinity GS over GDH. Unlike most members of the Methanosarcinaceae that have been found to be nonmotile,18 M. burtonii is motile by means of a single flagellum3 and possesses a bacterial-like Che chemotaxis system.4 Two flagellar proteins (FlaB) were more abundant in methanol-grown cells (Table 1), including one (Mbur_0346) that is located within a gene cluster containing chemotaxis genes. These changes may be linked to the need for M. burtonii to acquire substrates under challenging nutritional conditions.46 660


Increased levels of subunits of the carbon assimilation complexes ACDS and pyruvate synthase (pyruvate/ferredoxin oxidoreductase; POR) and several biosynthetic enzymes during growth on methanol may reflect a shift in carbon flow from energy generation to biosynthesis, including (but not limited to) 2-oxoglutarate production. However, the number of differentially abundant biosynthetic enzymes between methanoland TMA-grown cultures was low, suggesting that M. burtonii is performing de novo synthesis of amino acids in both media. MFM is a complex medium containing yeast extract, thus making amino acids and peptides available for direct uptake. As a methylotroph, M. burtonii is not expected to be able to use amino acids for energy generation, and it is not metabolically geared for their uptake; the genome lacks identifiable transporters for peptide uptake and only a few putative amino acid transporters have been identified.10 The proteomics data indicate that growth is mostly reliant upon endogenous amino acid production, irrespective of their presence in the surrounding medium. Overall, the increased abundance of citrate synthase, GS, ACDS, and POR in response to methanol indicates a global response of primary carbon and nitrogen metabolism to this substrate. Higher abundances of POR subunits, ThiC (Mbur_1494), and S-adenosylmethionine (SAM) synthetase in methanol-grown cells may be functionally linked. ThiC catalyzes a complex step in the synthesis of the pyrimidine moiety of thiamine pyrophosphate, which is an essential cofactor for POR. ThiC is a member of the Radical SAM protein family, which catalyze reactions that are initiated by a 5′adenosyl radical generated from the reductive cleavage of SAM.47,48 ACDS, ThiC and ferredoxin are iron-sulfur (4Fe-4S) cluster proteins, which may account for the higher levels of a 4Fe-4S cluster assembly protein in methanol-grown cells. Furthermore, LL-diaminopimelate aminotransferase and the AGAT family aminotransferase are pyridoxal phosphate (PLP)-dependent enzymes, which may also account for higher levels of the PLP synthase subunit in methanol-grown cells. A cobalamin synthesis protein, a sulfide dehydrogenase (SuDH) subunit, and a flavin mononucleotide binding (FMN)binding protein were more abundant in TMA-grown cells. The cobalamin synthesis protein is likely to be associated with the increased demand for coenzyme B (cobalamin). The roles of the other two proteins is less certain given the association of FMN-binding proteins with diverse electron transfer reactions49 and the multiple activities of SuDH.50 It is possibile that SuDH serves in a NADP recycling complex and assists the redox poise of the cell by using reduced ferredoxins (e.g., generated during methanogenesis) to furnish NADPH for ammonia assimilation. 1.4. Energy Conservation. Growth on methanol at 4 °C led to higher levels of a membrane-bound proton-translocating pyrophosphatase (PPase) subunit (Table 1). The membranebound PPase has been proposed to have concomitant roles in methanogenic archaea: disposal of cytoplasmically produced pyrophosphate (PPi), which thereby shifts PPi-generating reactions toward product formation (e.g., SAM synthetase); and salvaging free energy of PPi hydrolysis by proton translocation and the formation of a chemiosmotic gradient.51 Growth at 23 °C on methanol was also associated with the increased abundance of a Na+/H+ antiporter that has high identity to the Mrp complex of M. acetivorans (Table 1). The M. acetivorans Mrp complex is thought to conserve energy by utilizing a sodium gradient (high outside) across the cell membrane to antiport protons for use in ATP generation.52 It is likely that

Part II: The Effect of Different Methylated Growth Substrates the M. burtonii complex supplements the proton gradient established by Fpo (which was relatively unaffected by growth substrate) when cells are grown on methanol. It is possible that these energy-conservation mechanisms of M. burtonii are in greater demand during growth under nutrient depleted conditions, with methanol yielding less ∆G°′/mol substrate than TMA.15 Additionally, because reaction rates are lower at cold temperatures, the PPase may be favored at 4 °C due to its ability to drive biosynthetic reactions. 2. Stress Caused by Methanol. 2.1. Reactive Nonoxygen Species. SAM synthetase and the Radical SAM protein ThiC were both more abundant in methanol-grown cells (Table 1), and are likely to contribute to oxidative stress in M. burtonii. Adenosyl radicals generated by Radical SAM proteins are highly potent oxidizing species that have the potential to cause oxidative damage comparable to hydroxyl radicals53 (see Part I11). The bifunctional Fae/Hps (formaldehyde-activating enzyme/3-hexulose-6-phosphate synthase) protein was more abundant in methanol-grown cells (Table 1); this enzyme synthesizes ribulose-5-phosphate, and generates formaldehyde.54,55 Although formaldehyde is detoxified by Fae via a condensation reaction with H4SPT, if it is not efficiently converted to methylene-H4SPT, then this highly reactive compound would be toxic to cells. In Methanosarcina barkeri, RamA mediates the ATP-dependent reductive activation of the methylamine-specific MT corrinoid proteins (MttC, MtbC, MtmC).56 RamM is thought to catalyze the analogous reaction for methanol-specific MT corrinoid protein (MtaC), although this has not been experimentally verified. It is also not known if RamA and RamM can discriminate between methylamine and methanol MT corrinoid proteins. Similar to M. barkeri, the M. burtonii ramA gene is located in a TMA/DMA-specific MT gene cluster, and ramM is located in the methanol MT gene cluster (Figure 2). However, the abundance of both the M. burtonii RamA and RamM proteins was higher in methanol-grown cells (Table 1). One possible explanation for both RamA and RamM levels being higher in methanol-grown cells is that corrinoid proteins are prone to adventitious oxidation (and therefore inactivation) under these conditions, and higher Ram protein levels are required to counter this effect. Collectively these data indicate that growth on methanol favors metabolic pathways that are more likely to generate reactive nonoxygen species. This would account for the higher levels of universal stress (Usp) proteins (Table 1), which are thought to protect against DNA damage.57 A number of putative nucleic acid binding proteins were also more abundant in methanol grown cells (Table 1), and some of these may also act to protect DNA or RNA against oxidative damage. 2.2. Effect of Methanol on the Cell Surface. As a solvent, methanol has a toxic effect on the mechanical and permeable qualities of the cell membrane. Methanosarcina mazei adopts a “stress morphotype” in media containing methanol, including the increased aggregation of cells.58 While this specific response was not observed in M. burtonii, many secreted and cell surface proteins were more abundant in methanol-grown cells (Table 1). The cell surface proteins may assist in reinforcing the cell envelope against the solvent effects of methanol, and/or facilitate intercellular interactions. Many of these cell envelope proteins were also more abundant at 4 °C growth (see Part I12), which suggests a common protective mechanism against cold damage and methanol, and/or that solvent damage to the cell

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envelope is more likely to occur at low temperature, particularly as the substrate is metabolized more slowly. The gene for a YVTN/NHL protein (Mbur_1111) is located in a cluster that includes genes for many uncharacterized proteins that were all more abundant in methanol-grown cells. The gene for Mbur_1111 is adjacent to a gene that is annotated as a serpin (protease inhibitor; Mbur_1112). Serpins can regulate membrane-associated protein degradation, by associating with membrane proteases.59 Although inferred to have a role in cell-cell interactions,60 YVTN/NHL proteins may also be catalytic as this repeat domain (and the associated toroidal/beta-propeller structure) is associated with monooxygenase/dehydrogenases61,62 and proteases.63 As Mbur_1111 and Mbur_1112 are more abundant in methanol-grown cells they may function in the “quality control” of cell envelope proteins that are displaced or damaged by the action of methanol.

Acknowledgment. This work was supported by the Australian Research Council and U.S. Air Force Office of Scientific Research. Mass spectrometric results were obtained at the Bioanalytical Mass Spectrometry Facility within the Analytical Centre of the University of New South Wales. This work was undertaken using infrastructure provided by NSW Government coinvestment in the National Collaborative Research Infrastructure Scheme (NCRIS). Subsidized access to this facility is gratefully acknowledged. Note Added after ASAP Publication. This paper was published on the Web on Dec 1, 2009, with errors in the title of Table 2. The corrected version was reposted on Jan 7, 2010. References (1) Ferry, J. G.; Kastead, K. A. Methanogenesis. In Archaea. Molecular and Cellular Biology; Cavicchioli, R., Ed.; ASM Press: Washington, DC, 2007; pp 288-314. (2) Cavicchioli, R. Cold-adapted archaea. Nat. Rev. Microbiol. 2006, 4, 331–343. (3) Franzmann, P. D.; Springer, N.; Ludwig, W.; Conway De Macario, E.; Rohde, M. A methanogenic archaeon from Ace Lake, Antarctica: Methanococcoides burtonii sp. nov. System. Appl. Microbiol. 1992, 15, 573–581. (4) Goodchild, A.; Raftery, M.; Saunders, N. F. W.; Guilhaus, M.; Cavicchioli, R. Biology of the cold adapted archaeon, Methanococcoides burtonii determined by proteomics using liquid chromatography-tandem mass spectrometry. J. Proteome Res. 2004, 3, 1164–1176. (5) Goodchild, A.; Saunders, N. F. W.; Ertan, H.; Raftery, M.; Guilhaus, M.; Curmi, P. M. G.; Cavicchioli, R. A proteomic determination of cold adaptation in the Antarctic archaeon Methanococcoides burtonii. Mol. Microbiol. 2004, 53, 309–321. (6) Goodchild, A.; Raftery, M.; Saunders, N. F. W.; Guilhaus, M.; Cavicchioli, R. Cold adaptation of the Antarctic archaeon Methanococcoides burtonii assessed by proteomics using ICAT. J. Proteome Res. 2005, 4, 473–480. (7) Saunders, N. F. W.; Goodchild, A.; Raftery, M.; Guilhaus, M.; Curmi, P. M.; Cavicchioli, R. Predicted roles for Hypothetical proteins in the low-temperature expressed proteome of the Antarctic archaeon Methanococcoides burtonii. J. Proteome Res. 2005, 4, 464–472. (8) Saunders, N. F. W.; Ng, C.; Raftery, M.; Guilhaus, M.; Goodchild, A.; Cavicchioli, R. Proteomic and computational analysis of secreted proteins with type I signal peptides from the Antarctic archaeon Methanococcoides burtonii. J. Proteome Res. 2006, 5, 2457–2464. (9) Saunders, N. F. W.; Thomas, T.; Curmi, P. M.; Mattick, J. S.; Kuczek, E.; Slade, R.; Davis, J.; Franzmann, P. D.; Boone, D.; Rusterholtz, K.; Feldman, R.; Gates, C.; Bench, S.; Sowers, K.; Kadner, K.; Aerts, A.; Dehal, P.; Detter, C.; Glavina, T.; Lucas, S.; Richardson, P.; Larimer, F.; Hauser, L.; Land, M.; Cavicchioli, R. Mechanisms of thermal adaptation revealed from the genomes of the Antarctic archaea Methanogenium frigidum and Methanococcoides burtonii. Genome Res. 2003, 13, 1580–1588.


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