Quantitative Proteomic Analysis of Metabolic Regulation by Copper Ions in Methylococcus capsulatus (Bath)

Abstract

Copper ions switch the oxidation of methane by soluble methane monooxygenase to particulate methane monooxygenase in Methylococcus capsulatus (Bath). Toward understanding the change in cellular metabolism related to this transcriptional and metabolic switch, we have undertaken genomic sequencing and quantitative comparative analysis of the proteome in M. capsulatus (Bath) grown under different copper-to-biomass ratios by cleavable isotope-coded affinity tag technology. Of the 682 proteins identified, the expressions of 60 proteins were stimulated by at least 2-fold by copper ions; 68 proteins were down-regulated by 2-fold or more. The 60 proteins overexpressed included the methane and carbohydrate metabolic enzymes, while the 68 proteins suppressed were mainly responsible for cellular signaling processes, indicating a role of copper ions in the expression of the genes associated with the metabolism of the organism downstream of methane oxidation. The study has also provided a complete map of the C1 metabolism pathways in this methanotroph and clarified the interrelationships between them. Copper ions switch the oxidation of methane by soluble methane monooxygenase to particulate methane monooxygenase in Methylococcus capsulatus (Bath). Toward understanding the change in cellular metabolism related to this transcriptional and metabolic switch, we have undertaken genomic sequencing and quantitative comparative analysis of the proteome in M. capsulatus (Bath) grown under different copper-to-biomass ratios by cleavable isotope-coded affinity tag technology. Of the 682 proteins identified, the expressions of 60 proteins were stimulated by at least 2-fold by copper ions; 68 proteins were down-regulated by 2-fold or more. The 60 proteins overexpressed included the methane and carbohydrate metabolic enzymes, while the 68 proteins suppressed were mainly responsible for cellular signaling processes, indicating a role of copper ions in the expression of the genes associated with the metabolism of the organism downstream of methane oxidation. The study has also provided a complete map of the C1 metabolism pathways in this methanotroph and clarified the interrelationships between them. Methanotrophs are a unique group of Gram-negative bacteria that grow aerobically on methane and utilize methane as the sole source of carbon and energy. There has been considerable interest in methanotrophs over the past 30 years, since they can be used to produce single-cell bulk chemicals such as propylene oxide. The ability of these bacteria to co-oxidize a wide range of alkanes, alkenes, and substituted aliphatic compounds has been exploited in bioremediation processes, for example, in the degradation of key pollutants such as trichloroethylene in soil and groundwater. Methanotrophs also play an important role in the global methane cycle (1Hanson R.S. Hanson T.E. Microbiol. Rev. 1996; 60: 439-471Crossref PubMed Google Scholar, 2Trotsenko Y.A. Ivanova E.G. Doronina N.V. Mikrobiologiya. 2001; 70: 725-736Google Scholar). Methanotrophs are classified into three distinct types. Type I methanotrophs show disk-like intracellular membranes and use the RuMP (ribulose 5-phosphate) pathway for carbon assimilation. In contrast, Type II methanotrophs possess intracellular membranes in the cell periphery and use the serine pathway to degrade the formaldehyde produced. Methylococcus capsulatus (Bath) is classified as a Type X methanotroph (1Hanson R.S. Hanson T.E. Microbiol. Rev. 1996; 60: 439-471Crossref PubMed Google Scholar), since it shows the physiological properties of both Type I and II methanotrophs, but it develops Type I intracytoplasmic membranes. Two methane monooxygenases (MMO) 1The abbreviations used are: MMO, methane monooxygenase; pMMO, particulate methane monooxygenase; sMMO, soluble methane monooxygenase; BLAST, basic local alignment search tool; C1, one carbon; ICAT, isotope-coded affinity tag; cICAT, cleavable isotope-coded affinity tag; COG, clusters of orthologous groups of proteins; FBP, fructose 1,6-bisphosphate; H4MPT, tetrahydromethanopterin; KDPG, 2-keto-3-deoxy-6-phosphogluconate; L/H ratio, light-to-heavy ratio; ORF, open reading frame; RuMP, ribulose 5-phosphate; RuBP, ribulose bisphosphate; TMD, transmembrane domain; μLC-MS/MS, micro-capillary liquid chromatography tandem mass spectrometry; contig, group of overlapping clones. catalyze the methane oxidation process in methanotrophs, converting methane to methanol. All methanotrophs express a membrane-bound copper-containing particulate MMO (pMMO) (1Hanson R.S. Hanson T.E. Microbiol. Rev. 1996; 60: 439-471Crossref PubMed Google Scholar, 3Chan S.I. Chen K.H.-C. Yu S. S.-F. Chen C.-L. Kuo S. S.-J. Biochemistry. 2004; 43: 4421-4430Crossref PubMed Scopus (141) Google Scholar), while Type X and a few Type II methanotrophic bacteria are capable of producing a second, soluble form (soluble methane monooxygenase, sMMO). Copper ions are known to switch the methane oxidation from sMMO to pMMO. At low copper-to-biomass ratios, the cytoplasmic sMMO is the dominant MMO. When the cells are grown at high copper-to-biomass ratios, pMMO is expressed and produced instead in the plasma membrane. Because of the unique role of copper ions in regulating the switch between sMMO and pMMO, we have applied cICAT (cleavable isotope coded affinity tag) in combination with off-line two-dimensional LC-MS/MS to quantitatively compare the expression levels of the unique enzymes involved in the featured metabolic pathways under different copper environments. The ICAT technology has been developed for quantitative analysis of proteomic changes in response to cellular perturbations with wide dynamic range and quantitation accuracy (4Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4362) Google Scholar, 5Aebersold R. Mann M. Nature. 2003; 422: 198-207Crossref PubMed Scopus (5639) Google Scholar). More recently, the cICAT reagent has also been developed to eliminate chromatographic isotope effects caused by hydrogen and deuterium (6Li J. Steen H. Gygi S.P. Mol. Cell. Proteomics. 2003; 2: 1198-1204Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Two groups (the National High-Throughput Genome Sequencing Center at the National Yang-Ming University in Taiwan and the Institute for Genomic Research (Rockville, MD) (www.tigr.org/tdb/mdb/mdbinprogress.html)) have been independently making substantial progress toward completing the sequencing of the genome of this methanotroph. These data, although incomplete, have been indispensable to the present proteomic study. Sample Preparation and cICAT Labeling—The culturing and growth of M. capsulatus (Bath) and the separation of the cellular materials into cytoplasmic and membrane fractions were carried out following the procedure developed recently (7Yu S. S.-F. Chen K. H.-C. Tseng M. Y.-H. Wang Y.-S. Tseng C.-F. Chen Y.-J. Huang D.-S. Chan S.-I. J. Bacteriol. 2003; 185: 5915-5924Crossref PubMed Scopus (97) Google Scholar). Four fractions of proteins were obtained: two cytosolic fractions and two intracellular membrane fractions corresponding to the protein samples derived from cells grown in 0 and 30 μm copper. All samples were lyophilized for quantitation. Protein samples were prepared at a concentration of 1 mg/ml using the extraction buffer (0.3% SDS, 50 mm Tris, pH 8.3, 5 mm EDTA, and 6 m urea) by weighing out samples of the lyophilized powder. The protein concentration was verified by Bio-Rad protein assay. Labeling of the proteins by the cICAT reagents and following procedures before MS analysis were carried out as described in the literature (8Yi E.C. Goodlett D. Current Protocol in Protein Science Online. John Wiley & Sons, Inc., Edison, NJ2004Google Scholar). Peptide Separation and Purification—The peptides were separated by cation exchange chromatography using a 4.6 × 200-mm polysulfoethyl A column (5-μm particles, 300-A pore size; Poly LC, Columbia, MD) at a flow rate of 200 μl/min. Peptides were eluted by a gradient of 0-25% Buffer B over 30 min, followed by 25-100% Buffer B over 20 min (Buffer A: 5 mm K2HPO4, 25% CH3CN, pH = 3.0; Buffer B: 5 mm K2HPO4, 25% CH3CN, 350 mm KCl, pH = 3.0). The elution profile of the cation exchange chromatography determined which fractions were to be analyzed further, and each were individually processed over avidin cartridges (Applied Biosystems), and the affinity tags were cleaved according to the manufacturer's protocol (cICAT kit for protein labeling; Applied Biosystems) to isolate the labeled Cys-containing peptides for micro-capillary liquid chromatography tandem mass spectrometry (μLC-MS/MS) analysis. μLC-MS/MS Analysis—Samples were loaded using an auto-sampler and sequentially analyzed by μLC-MS/MS. Injections were made on a 1.5 cm × 100 μm trapping and 12 cm x 75 μm separation column packed in-house (Magic C18; Micromass BioResources, Auburn, CA). Peptides were eluted with a linear gradient of 5-40% Buffer B over 180 min at an elution rate of ∼200 nl/min (Buffer A: 0.1% formic acid in H2O; Buffer B: 0.1% formic acid in acetonitrile). A Hewlett Packard 1100 solvent delivery system with flow splitting (Hewlett Packard, Palo Alto, CA) was used. An LCQ-Deca ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) with an in-house built microspray device was used for all analyses. Peptide fragmentation by collision-induced dissociation was carried out in an automated fashion using the dynamic-exclusion option, and the resultant MS/MS spectra were recorded. The interpretation of the MS/MS data were finally submitted to a suit of software tools for automated data base searching and statistical interpretation of the search results. Data Analysis and Bioinformatics—Genomic sequencing of M. capsulatus (Bath) was performed by the National High-Throughput Genome Sequencing Center at the National Yang-Ming University. The entire shotgun sequencing is not finished; however, we have obtained both the 4× and 8× coverage contigs. EMBOSS 2.8.0 (9Rice P. Longden I. Bleasby A. Trends Genet. 2000; 16: 276-277Abstract Full Text Full Text PDF PubMed Scopus (6541) Google Scholar), NCBI BLAST 2.2.6 (10Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar), and HMMER 2.3.2 (11Eddy S.R. Bioinformatics. 1998; 14: 755-763Crossref PubMed Scopus (4105) Google Scholar) were installed on FreeBSD 4.8 RELEASE with one Intel® Pentium® 4 processor operating in the batch mode. Open reading frames (ORFs) were predicted and translated into protein sequences by the GETORF function of the EMBOSS suite, and their homologues protein names were searched against the Swiss-Prot, TrEMBL, and the NCBI non-redundant data bases with the expectation value of 1E-05 as the criterion. 5485 protein homologues were predicted. Since the homologues of the ORFs were not exactly the ones that gave the best similarity (for example, some hypothetical proteins were predicted by genomic sequencing), all the entries were checked manually. Automated data base searching using SEQUEST (12Eng J.K. McCormack A.L. Yates II I J. PubMed Scopus Google Scholar) software was performed to and protein for each MS/MS MS/MS spectra were searched against the M. capsulatus (Bath) genomic sequencing data SEQUEST search for were to with a for and for mass peptides were to the search results. were by Peptide and peptides that Peptide were analyzed by Protein to open reading frames A. Aebersold R. PubMed Scopus Google Scholar, A. Aebersold R. 2003; PubMed Scopus Google Scholar). The to ratios from the were and by Zhang H. Aebersold R. 2003; PubMed Scopus Google Scholar) Analysis on Protein function was performed with the NCBI on the and each protein was into the group Nucleic Acids Res. 2001; PubMed Scopus Google Scholar). Protein were classified by HMMER with the Protein were analyzed by the function of EMBOSS 2.8.0 with the data base A. P. A. Nucleic Acids Res. 2004; PubMed Google Scholar). Protein were predicted by the K. M. Genet. PubMed Scopus Google Scholar), and the of transmembrane was predicted by the function of EMBOSS 2.8.0 B. P. J. Mol. PubMed Scopus Google Scholar). of Copper for the to study (7Yu S. S.-F. Chen K. H.-C. Tseng M. Y.-H. Wang Y.-S. Tseng C.-F. Chen Y.-J. Huang D.-S. Chan S.-I. J. Bacteriol. 2003; 185: 5915-5924Crossref PubMed Scopus (97) Google Scholar), 30 μm is the copper concentration for the growth and of cells of M. capsulatus (Bath). the present the growth in the was by a membrane (7Yu S. S.-F. Chen K. H.-C. Tseng M. Y.-H. Wang Y.-S. Tseng C.-F. Chen Y.-J. Huang D.-S. Chan S.-I. J. Bacteriol. 2003; 185: 5915-5924Crossref PubMed Scopus (97) Google Scholar), and the cells were grown at a copper ion concentration of 30 μm and in the that a was between the copper in the and the copper concentration in the growth the cells grow at of copper the pMMO in the membranes was not and not all the of copper ions and the of copper ions were not used as copper ions be by the cells (7Yu S. S.-F. Chen K. H.-C. Tseng M. Y.-H. Wang Y.-S. Tseng C.-F. Chen Y.-J. Huang D.-S. Chan S.-I. J. Bacteriol. 2003; 185: 5915-5924Crossref PubMed Scopus (97) Google Scholar). 682 in expressed in the and of copper 0 and 30 were labeled with and cICAT In a of 682 proteins of M. capsulatus (Bath) were with at least one of high were from the cytosolic and from the membrane it was to the change in expression levels of of the 682 proteins from the L/H mass ratios of the protein of the proteins has to known proteins in the and data entries of the from M. capsulatus (Bath). The proteins show low or to proteins in the data base and are unique proteins expressed in M. capsulatus (Bath) or that have not been the of the 682 the ORFs were analyzed by different and membrane proteins in M. capsulatus the was used. were also predicted by the function of The analysis that the proteins were mainly in the soluble Of the proteins predicted to be in the membrane proteins were predicted to two or transmembrane The of cytosolic proteins with two or transmembrane was indicating that was some of the cytosolic with membrane The cytosolic proteins were and were Gram-negative membrane These are in from pMMO proteins in the membrane included the and of = and = and and = the and the acid protein L/H = The L/H = L/H = the and the hypothetical protein L/H = were also with a of transmembrane predicted. All unique protein sequences were analyzed by the NCBI proteins all the sMMO and pMMO not be classified in this on as the data and has been and genomic sequencing of methanotrophs the present study. is sequencing the genome of M. capsulatus (Bath) the data from this source is and for the proteomic study undertaken the expression levels 2-fold for of the proteins in the present cICAT proteomic the of the proteins were to be different is from the of the proteins that the metabolism of the cell be by copper ion concentration The entire metabolic the but the of the metabolic pathways are at 30 μm the 60 proteins with L/H of be classified as enzymes involved in the of cell and ion and in the oxidation of methane to carbon are pMMO, = = and = pMMO and is the protein produced at 30 μm copper as by (7Yu S. S.-F. Chen K. H.-C. Tseng M. Y.-H. Wang Y.-S. Tseng C.-F. Chen Y.-J. Huang D.-S. Chan S.-I. J. Bacteriol. 2003; 185: 5915-5924Crossref PubMed Scopus (97) Google Scholar). cICAT on the of for the in pMMO is There is in one in and are three in the was = and we were in pMMO from the transmembrane of and The L/H was indicating that pMMO was overexpressed by at 30 μm copper to the that was in the proteome derived from cells grown as from (7Yu S. S.-F. Chen K. H.-C. Tseng M. Y.-H. Wang Y.-S. Tseng C.-F. Chen Y.-J. Huang D.-S. Chan S.-I. J. Bacteriol. 2003; 185: 5915-5924Crossref PubMed Scopus (97) Google Scholar). are to by the cICAT In the extraction and of transmembrane peptides a The = was protein that was by copper. was by BLAST searching as = which is involved in of a wide of The of proteins the high of formaldehyde produced at 30 μm copper from methane oxidation by pMMO, to the high levels of pMMO in the membranes as as the of methane in the intracytoplasmic membranes. The = the in the pathway of methane was also in the membrane Since formaldehyde is to the these enzymes are to the formaldehyde the of these enzymes to be the genome sequencing data we have all the genes involved in the acid of the enzymes, were in the cICAT study. = and = on was also identified, with a different L/H of = = and (the L/H = were stimulated at 30 μm copper. has been that M. capsulatus (Bath) a complete acid cycle to the of The of however, the has been = These that Type X some metabolic properties of both Type I and Type II Type II methanotrophs use the serine pathway as C1 with a complete acid while Type I methanotrophs not (1Hanson R.S. Hanson T.E. Microbiol. Rev. 1996; 60: 439-471Crossref PubMed Google Scholar, The of Scholar). is that some cell and related proteins such as = = = = and = are stimulated at the copper ion the cell to the intracytoplasmic it is that the of cell is bacteria with pMMO also have growth with sMMO (1Hanson R.S. Hanson T.E. Microbiol. Rev. 1996; 60: 439-471Crossref PubMed Google Scholar). The high copper is with the of the M. capsulatus (Bath). Four ion proteins are also enzymes the source for = the in = the involved in the of and = which is involved in = was at 30 μm copper in both the cytosolic and membrane is an in but the is also known J. J. J. Biochemistry. 2000; PubMed Scopus Google Scholar). A BLAST search that the is have this and is to from indicating the of and S. I. this is the Since the of expression of the is at the copper we that as to the the cell for by the pMMO, at the high levels of pMMO produced at 30 μm copper. at 30 μm are three protein sequences with similarity in the data base and hypothetical The by similarity search are in An A was in open reading = and = three and one and their are these proteins be their expressions are stimulated at the copper with at 0 μm we the proteins that were expressed at levels in the of copper. The 68 proteins with L/H to different of the cell the proteins with to be proteins related to protein and and In enzymes related to carbohydrate metabolism were sMMO with L/H = and was also identified, but L/H not be and = The is responsible for the of methane oxidation and has been J. 2001; PubMed Scopus Google Scholar). Since M. capsulatus (Bath) not on as the carbon and are the ability of M. capsulatus (Bath) to grow on the function of be involved in or Two of the and in the sMMO were also = is a involved in the of the sMMO while = is a to function as a copper R. J. 2003; PubMed Scopus Google Scholar). change was in the expression of the cell signaling and proteins were expressed the cells were grown the transcriptional protein = protein = expression = and the protein = Two proteins that cell were identified, the cell = and the cell = was the = with which peptides by to form in with such as proteins against proteins such as = involved in and the = were also Two proteins responsible for were key enzymes involved in metabolism were with at 0 μm copper. The = is responsible for the of to formic acid in the pathway A. PubMed Scopus Google Scholar). I = is involved in the of the of which is a C1 in some and A. PubMed Scopus Google Scholar). The = = and = are enzymes involved in the and the of enzymes were L/H = L/H = and = = was BLAST searched as the in J. R. S. A. 1996; PubMed Scopus Google Scholar), but the role of this in bacteria has not been and to be in have also the protein = the cells were grown at 0 μm copper The of this protein has been to be by M. capsulatus (Bath) into the growth with the of some copper function A. Microbiol. 2001; PubMed Scopus Google Scholar), but this protein was not in copper-containing environments. = is to play a role in Gram-negative or not copper also the membrane is but this an we proteins with BLAST as as hypothetical proteins expression levels were copper to the growth The one is = which of one one A and = also shows three and one be related to the organism is grown at low copper-to-biomass of Protein expression of the two are distinct (7Yu S. S.-F. Chen K. H.-C. Tseng M. Y.-H. Wang Y.-S. Tseng C.-F. Chen Y.-J. Huang D.-S. Chan S.-I. J. Bacteriol. 2003; 185: 5915-5924Crossref PubMed Scopus (97) Google Scholar). The of the present proteomic study this and the metabolic and physiological The expressed levels of the carbon enzymes in the 30 μm copper the expressions of = = = = = the in the RuMP the of the acid the serine and the in of serine The of these pathways is The of formaldehyde to be by which MMO is used by the organism for methane oxidation. have a protein = that to the of these genes and or their to M. H. J. Bacteriol. PubMed Google Scholar). The capsulatus (Bath) methane as carbon The oxidation protein L/H = the of which is between the genes for the two be involved in of the in the D.J. PubMed Scopus Google Scholar). is the important metabolic in the metabolism of these methanotrophs, metabolic oxidation of the RuMP the serine and the all from this key enzymes in the RuMP pathway were of the RuMP = In the metabolism of methanotrophs, are two of both the or and the or of the RuMP pathway The of Scholar), are have both the = and the = as as all the enzymes involved in was in and of the genomic both the and are although it is that methanotrophs use the = and = are key enzymes of the but M. capsulatus (Bath) not to be to grow Microbiol. PubMed Scopus Google Scholar). The pathway enzymes be in the of the RuMP is it M. capsulatus (Bath) both in the RuMP is that the RuMP pathway is the of the pathway Microbiol. Rev. PubMed Google Scholar). In methanotrophs, the carbon source is methane the RuMP pathway the for carbohydrate All are to and the acid cycle is used to complete the we can that is in the entire carbohydrate metabolism is that M. capsulatus (Bath) has an serine and on this it has been classified as a Type X methanotroph (1Hanson R.S. Hanson T.E. Microbiol. Rev. 1996; 60: 439-471Crossref PubMed Google Scholar, The of Scholar). we have all the genes of the serine pathway and all the proteins as in the present cICAT There is that the serine pathway is to the pathway The of Scholar), since be by the of and by = to which is to by = The for the is downstream of the of all enzymes of the pathway were is the C1 pathway in since in A. PubMed Scopus Google Scholar, J. Bacteriol. 1999; PubMed Google Scholar, Chen A. J. Bacteriol. 2003; 185: PubMed Scopus Google Scholar). to genomic the = and for A and and = is to the of the S. T.L. 1999; PubMed Scopus Google Scholar), while the genes of the = protein searched as and = form in the Since the was a and Microbiol. Rev. PubMed Google Scholar), compounds have been to play a role in Nature. 2001; PubMed Scopus Google Scholar, W. J. K. Nature. 2001; PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar), and has been considerable interest in that utilize C1 compounds in The present genomic and proteomic study has to the metabolic pathways in methanotrophic carbon assimilation. are to for a of with