Proteome Analysis of Halobacterium sp. NRC-1 Facilitated by the Biomodule Analysis Tool BMSorter
2006; Elsevier BV; Volume: 5; Issue: 6 Linguagem: Inglês
10.1074/mcp.m500367-mcp200
ISSN1535-9484
AutoresRuei-Chi Richie Gan, Eugene C. Yi, Yulun Chiu, Hookeun Lee, Yu-chieh P. Kao, Timothy H. Wu, Ruedi Aebersold, David R. Goodlett, Wailap Victor Ng,
Tópico(s)Microbial Community Ecology and Physiology
ResumoTo better understand the extremely halophilic archaeon Halobacterium species NRC-1, we analyzed its soluble proteome by two-dimensional liquid chromatography coupled to electrospray ionization tandem mass spectrometry. A total of 888 unique proteins were identified with a ProteinProphet probability (P) between 0.9 and 1.0. To evaluate the biochemical activities of the organism, the proteomic data were subjected to a biological network analysis using our BMSorter software. This allowed us to examine the proteins expressed in different biomodules and study the interactions between pertinent biomodules. Interestingly an integrated analysis of the enzymes in the amino acid metabolism and citrate cycle networks suggested that up to eight amino acids may be converted to oxaloacetate, fumarate, or oxoglutarate in the citrate cycle for energy production. In addition, glutamate and aspartate may be interconverted from other amino acids or synthesized from citrate cycle intermediates to meet the high demand for the acidic amino acids that are required to build the highly acidic proteome of the organism. Thus this study demonstrated that proteome analysis can provide useful information and help systems analyses of organisms. To better understand the extremely halophilic archaeon Halobacterium species NRC-1, we analyzed its soluble proteome by two-dimensional liquid chromatography coupled to electrospray ionization tandem mass spectrometry. A total of 888 unique proteins were identified with a ProteinProphet probability (P) between 0.9 and 1.0. To evaluate the biochemical activities of the organism, the proteomic data were subjected to a biological network analysis using our BMSorter software. This allowed us to examine the proteins expressed in different biomodules and study the interactions between pertinent biomodules. Interestingly an integrated analysis of the enzymes in the amino acid metabolism and citrate cycle networks suggested that up to eight amino acids may be converted to oxaloacetate, fumarate, or oxoglutarate in the citrate cycle for energy production. In addition, glutamate and aspartate may be interconverted from other amino acids or synthesized from citrate cycle intermediates to meet the high demand for the acidic amino acids that are required to build the highly acidic proteome of the organism. Thus this study demonstrated that proteome analysis can provide useful information and help systems analyses of organisms. Halobacterium species NRC-1 is an extremely halophilic archaeon containing a highly acidic proteome with a median pI of 4.9, a property that is essential to the maintenance of the solubility and function of the proteins in a high salinity environment of about 5 m salts (1Kennedy S.P. Ng W.V. Salzberg S.L. Hood L. DasSarma S. Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence.Genome Res. 2001; 11: 1641-1650Crossref PubMed Scopus (259) Google Scholar, 2Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Crossref PubMed Scopus (600) Google Scholar). Genome sequence analysis has revealed 2,630 putative protein-coding genes in the 2,571,010-bp genome (2Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Crossref PubMed Scopus (600) Google Scholar). Among the predicted proteins, 1,658 can be matched to sequences in public databases. Of the matches, 1,067 are proteins of known or predicted function, and 591 are proteins of unknown function. The possession of a relatively small and completely sequenced genome, the availability of a full arsenal of genetic manipulation tools, and the relative ease of culture make Halobacterium sp. NRC-1 an attractive systems biology model organism of the domain Archaea (3DasSarma S. Robb F.T. Place A.R. Sowers K.R. Schreier H.J. Archaea: a Laboratory Manual—Halophiles. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1995: 197-208Google Scholar, 4Peck R.F. DasSarma S. Krebs M.P. Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker.Mol. Microbiol. 2000; 35: 667-676Crossref PubMed Scopus (147) Google Scholar, 5Cline S.W. Lam W.L. Charlebois R.L. Schalkwyk L.C. Doolittle W.F. Transformation methods for halophilic archaebacteria.Can. J. Microbiol. 1989; 35: 148-152Crossref PubMed Scopus (229) Google Scholar, 6Cline S.W. Doolittle W.F. Efficient transfection of the archaebacterium Halobacterium halobium.J. Bacteriol. 1987; 169: 1341-1344Crossref PubMed Scopus (102) Google Scholar). The genomes of Halobacterium species are extremely unstable (7Sapienza C. Rose M.R. Doolittle W.F. High-frequency genomic rearrangements involving archaebacterial repeat sequence elements.Nature. 1982; 299: 182-185Crossref PubMed Scopus (58) Google Scholar, 8Sapienza C. Doolittle W.F. Unusual physical organization of the Halobacterium genome.Nature. 1982; 295: 384-389Crossref PubMed Scopus (60) Google Scholar, 9Pfeifer F. Weidinger G. Goebel W. Genetic variability in Halobacterium halobium.J. Bacteriol. 1981; 145: 375-381Crossref PubMed Google Scholar). Early studies of Halobacterium sp. NRC-1 (also known as Halobacterium halobium) and the closely related Halobacterium salinarium discovered unusually high spontaneous mutation frequencies of 0.01% for the production of bacteriorhodopsin- or bacterioruberin-deficient phenotypes and a more striking 1% for partial or total gas vesicle-deficient phenotypes. The species is also noteworthy for the large number of insertion sequence elements that are harbored in this unstable genome. Molecular genetic analysis of the bacteriorhodopsin- and gas vesicle-deficient mutants established a relationship between transposable insertion sequence-mediated insertional inactivation or deletions of structural or regulatory genes and the high mutant rates (10DasSarma S. Mechanisms of genetic variability in Halobacterium halobium: the purple membrane and gas vesicle mutations.Can. J. Microbiol. 1989; 35: 65-72Crossref PubMed Scopus (40) Google Scholar, 11DasSarma S. RajBhandary U.L. Khorana H.G. High-frequency spontaneous mutation in the bacterio-opsin gene in Halobacterium halobium is mediated by transposable elements.Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2201-2205Crossref PubMed Scopus (66) Google Scholar, 12Jones J.G. Hackett N.R. Halladay J.T. Scothorn D.J. Yang C.F. Ng W.L. DasSarma S. 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A. 1982; 79: 7268-7272Crossref PubMed Scopus (45) Google Scholar). Upon the completion of the genome sequence, DNA analysis revealed the presence of 91 copies of insertion sequence elements belonging to 12 families in the Halobacterium sp. NRC-1 genome (2Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Crossref PubMed Scopus (600) Google Scholar). Mass spectrometry is a powerful technology for protein identification in the postgenomic era. Our previous shotgun peptide sequencing analysis of the Halobacterium sp. NRC-1 membrane and soluble proteomes identified a total of 426 unique proteins representing approximately one-fifth of the predicted proteome (2Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Crossref PubMed Scopus (600) Google Scholar, 17Goo Y.A. Yi E.C. Baliga N.S. Tao W.A. Pan M. Aebersold R. Goodlett D.R. Hood L. Ng W.V. Proteomic analysis of an extreme halophilic archaeon, Halobacterium sp. NRC-1.Mol. Cell. Proteomics. 2003; 2: 506-524Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Among these, 232 were identified predominantly in the soluble fraction, 165 were in the membrane fraction, and 29 were in both fractions. Metabolic reconstruction found 103 of the identified proteins could be matched to enzymes in 52 metabolic pathways found in the Kyoto Encyclopedia of Genes and Genomes (KEGG) 1The abbreviations used are: KEGG, Kyoto Encyclopedia of Genes and Genomes; p, PeptideProphet probability; P, ProteinProphet probability; CGI, common gateway interface; SIF, simple interaction format; TBP, TATA box-binding protein; TFB, transcription factor B; aa, amino acids. database (www.genome.ad.jp). Here we report the systems analysis of the Halobacterium sp. NRC-1 soluble proteome identified by two-dimensional liquid chromatography coupled with tandem mass spectrometry. The analysis was facilitated by the BMSorter 2The software BMSorter, HalKGMLtoSIF.java, and HalnoaFactory.java are available upon request to [email protected] tool that incorporates the protein identification results into biological networks. In addition to being able to document the proteins identified in this study, we demonstrate the power of integrative analysis of proteomic data in the analysis of biological networks. One liter of Halobacterium sp. NRC-1 (ATCC 700922) cells was grown in Halobacterium medium to early log phase (A600 = 1) at 37 °C (17Goo Y.A. Yi E.C. Baliga N.S. Tao W.A. Pan M. Aebersold R. Goodlett D.R. Hood L. Ng W.V. Proteomic analysis of an extreme halophilic archaeon, Halobacterium sp. NRC-1.Mol. Cell. Proteomics. 2003; 2: 506-524Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 18Oesterhelt D. Stoeckenius W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane.Methods Enzymol. 1974; 31: 667-678Crossref PubMed Scopus (1594) Google Scholar) and harvested by centrifugation at 5,500 × g for 10 min at 4 °C. The cell pellet was resuspended in a total of 25 ml of basal salt solution containing 1 mm PMSF and 0.5 mg each of DNase I and RNase A. The cells were transferred to a dialysis tube (Spectrapor®; membrane molecular weight cutoff; 3,500; Spectrum Laboratories, Inc.) and lysed by osmotic shock by dialysis against four changes of 4 liters of deionized water at 4 °C over a total of 2 days. Cell debris were removed by centrifugation at 10,000 × g for 30 min at 4 °C. Membrane and insoluble materials were sedimented by ultracentrifugation at 53,000 × g for 16 h at 4 °C. The supernatant containing the soluble proteins was aliquoted into 1.5-ml microcentrifuge tubes and stored at −20 °C. An aliquot of 3 mg of soluble proteins was digested with 60 μg of sequencing grade modified trypsin (Promega, Madison, WI) in a total volume of 3 ml of a solution containing 50 mm ammonium bicarbonate (pH 8.3) at 37 °C overnight. The resulting peptides were fractionated by strong cation exchange chromatography as described previously (19Han D.K. Eng J. Zhou H. Aebersold R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.Nat. Biotechnol. 2001; 19: 946-951Crossref PubMed Scopus (829) Google Scholar). The fractionated peptides were desalted using 96-well format spin columns containing silica C18 matrix (Nested Group, Southborough, MA) according to the following procedure. First the matrix was washed twice by filling each well with 200 μl of 0.4% acetic acid and centrifuged at 770 × g for 2 min. After loading the peptide samples (∼200 μl/well), the plate was incubated at room temperature for 30 min and then centrifuged as above to remove the buffer. The reversed phase C18-bound peptides were washed three times with 200 μl of 0.4% acetic acid and eluted with 200 μl of a solution containing 3 volumes of acetonitrile and 1 volume of 0.4% acetic acid. The eluents were vacuum-dried, and each peptide samples was resuspended in 10 μl of 0.4% acetic acid before analysis by LC-MS/MS. The desalted tryptic peptides were analyzed using a LCQ-DECA ion trap tandem mass spectrometer (Thermo Finnegan, San Jose, CA) coupled with a C18 trap ESI-emitter/micro-liquid chromatography column as described previously (20Yi E.C. Marelli M. Lee H. Purvine S.O. Aebersold R. Aitchison J.D. Goodlett D.R. Approaching complete peroxisome characterization by gas-phase fractionation.Electrophoresis. 2002; 23: 3205-3216Crossref PubMed Scopus (176) Google Scholar). Tandem mass spectra were analyzed using the SEQUEST (21Eng J.K. McCormack A.L. Yates III, J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5472) Google Scholar) algorithm to search against the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) Halobacterium sp. NRC-1 proteome database (2Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Crossref PubMed Scopus (600) Google Scholar). The searches were performed with a peptide mass tolerance of 3 daltons without enzyme specification. The other parameters were left as default. The SEQUEST outputs were consolidated to a single hypertext file and further analyzed using the proteomic data analysis pipeline (Institute for Systems Biology; www.systemsbiology.org). As part of this pipeline, the programs PeptideProphet and ProteinProphet (22Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3912) Google Scholar, 23Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3655) Google Scholar) assign probability values to each peptide and protein identification, respectively, that indicate the likelihood that the respective analyte has been identified correctly. BMSorter is a bioinformatic tool developed to facilitate systems analysis of proteins detected by tandem mass spectrometry. It is a set of common gateway interface (CGI) scripts written in Perl language to provide an interface to access the results of analyses via web browser. The scripts require an environment with an Apache web server and Perl module: Bioperl1.4, CGI-3.04, and GD-2.16. BMSorter constructed a list of expressed proteins with interactions between the proteins. It acquired the protein list with identification probabilities from the ProteinProphet output file as well as the pathway information in the form of a template of interactions from the KEGG database. Cross-reference between pathway maps from KEGG PATHWAYS database and enzyme/gene information from KEGG LIGAND database implemented the integration between proteins and interactions. The tool set organized the proteomic data and KEGG pathway information in a summary table. Each row of the table is the summary of a pathway (or biomodule) containing the total number of reactions in the reference pathway, number of Halobacterium sp. NRC-1-specific reactions, percentage of NRC-1 reactions, number of identified proteins with ProteinProphet probabilities (P) ≥0.9, and percentage of identified proteins plus the hyperlinks to the pathway protein list tables and pathway maps with the ProteinProphet identification probabilities indicated. The publicly available Cytoscape tool (www.cytoscape.org) was used to display the protein identification information on amino acid metabolisms and the citrate cycle pathways in terms of the enzyme-metabolite interaction networks (24Shannon P. Markiel A. Ozier O. Baliga N.S. Wang J.T. Ramage D. Amin N. Schwikowski B. Ideker T. Cytoscape: a software environment for integrated models of biomolecular interaction networks.Genome Res. 2003; 13: 2498-2504Crossref PubMed Scopus (26615) Google Scholar). The Java programs HalKGMLtoSIF.java and HalnoaFactory.java were developed to process the KEGG pathways KEGG mark-up language (KGML) files and proteomic data, respectively. The HalKGMLtoSIF.java program was used to parse the extensible markup language (xml) structure of the KGML files to generate the Cytoscape simple interaction format (SIF) and node attribute files. The HalnoaFactory.java was used to define the node properties (protein names and ProteinProphet probability values) in node attribute files. Three milligrams of Halobacterium sp. NRC-1 trypsin-digested soluble protein were separated into 100 fractions by strong cation exchange HPLC. The peptides contained in most fractions were desalted in a 96-well format C18 plate. The strong cation exchange fractions 11–85 were either analyzed as single fractions or pools of up to three consecutive fractions by LC-MS/MS. A total of 46 samples were thus analyzed of which 16 were analyzed twice, resulting in 62 LC-MS/MS datasets. The SEQUEST output data were analyzed with the PeptideProphet and ProteinProphet software to evaluate and assign, respectively, the peptide and protein identification probabilities. A total of 888 proteins were identified with P between 0.9 and 1.0. Among these were 562 proteins with annotated functions, 189 conserved hypothetical proteins, and 137 hypothetical proteins of unknown functions (Supplemental Tables I and II). The distributions of probabilities and functional categories of identified proteins are summarized in Fig. 1. A total of 681 and 147 proteins were identified with a probability of 1.0 and 0.99, respectively. The estimated number of false positive identifications of N proteins with a probability value of P is equal to N × (1.0 − P). The total number of false positive protein identifications was ∼5. The identified proteins were assigned to appropriate biomodules by the BMSorter tool (Fig. 2). From the proteomic data and the information on enzymes, metabolites, and chemical reactions available from the KEGG database, BMSorter generated a table containing the number of reactions in the reference pathways and their corresponding Halobacterium sp. NRC-1 pathways, the number of predicted and mass spectrometric identified proteins in each NRC-1 pathway, the percentages of NRC-1 reactions, and the percentages of identified NRC-1 proteins together with hyperlinks to the pathway maps and lists of enzymes in each biomodule (Table I). A total of 297 proteins (P > 0.9) were matched to 76 biomodules in the KEGG database. The proteins identified in representative biomodules or functional categories are discussed in detail below.Table ISummary of the percentage of mass spectrometry-identified Halobacterium sp. NRC-1 proteins, percentage of NRC-1-specific reactions, number of MS-identified proteins, numbers of NRC-1 predicted proteins and reactions, and number of reactions in the reference map in each KEGG pathway/protein complex/cellular functionView Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Genome analysis has revealed five genes encoding three DNA polymerase types in Halobacterium sp. NRC-1 including two family B polymerases (polB1 on the chromosome and polB2 on the minichromosome pNRC200), a bacteriophage-like family A polymerase (polC), and the heterodimeric family D polymerase (polA1 and polA2) (2Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Crossref PubMed Scopus (600) Google Scholar). Among these only PolA2 and PolB1 were detected in this study. In addition, DNA polymerase sliding clamp (Pcn) protein, clamp loader (RfcA, RfcB, and RfcC subunits), DNA topoisomerase VI subunits A (Top6A) and B (Top6B), DNA topoisomerase I (TopA), DNA gyrase subunits A (GyrA) and B (GyrB), DNA helicase (Hel), and replication protein A (Rpa) involved in single strand DNA binding were also identified (Supplemental Table I). The transcription of Halobacterium sp. NRC-1 genes is driven by a eukaryotic RNA polymerase II-like system. It consists of 12 subunits (A, B′, B‴, C, D, E′, E‴, H, K, L, M, and N) encoded by genes located at six loci. Except for the H and K subunits, all the subunits were identified by tandem mass spectrometry with P ≥ 0.9. Transcription initiation in Archaea requires the binding of the TATA box-binding protein (TBP) and transcription factor B (TFB) to the promoter region (25Bell S.D. Magill C.P. Jackson S.P. Basal and regulated transcription in Archaea.Biochem. Soc. Trans. 2001; 29: 392-395Crossref PubMed Scopus (56) Google Scholar, 26Kosa P.F. Ghosh G. Decker B.S. Sigler P.B. The 2.1-Å crystal structure of an archaeal preinitiation complex: TATA-box-binding protein ranscription factor (II)B core/TATA-box.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6042-6047Crossref PubMed Scopus (145) Google Scholar, 27Soppa J. Link T.A. The TATA-box-binding protein (TBP) of Halobacterium salinarum. Cloning of the tbp gene, heterologous production of TBP and folding of TBP into a native conformation.Eur. J. Biochem. 1997; 249: 318-324Crossref PubMed Scopus (12) Google Scholar). Genome analysis identified the presence of six tbp and seven tfp genes (2Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Crossref PubMed Scopus (600) Google Scholar, 28Baliga N.S. Goo Y.A. Ng W.V. Hood L. Daniels C.J. DasSarma S. Is gene expression in Halobacterium NRC-1 regulated by multiple TBP and TFB transcription factors?.Mol. Microbiol. 2000; 36: 1184-1185Crossref PubMed Scopus (76) Google Scholar, 29Ng W.V. Ciufo S.A. Smith T.M. Bumgarner R.E. Baskin D. Faust J. Hall B. Loretz C. Seto J. Slagel J. Hood L. DasSarma S. Snapshot of a large dynamic replicon in a halophilic archaeon: megaplasmid or minichromosome?.Genome Res. 1998; 8: 1131-1141Crossref PubMed Scopus (88) Google Scholar). Only two TATA-box binding proteins and one Tfb protein, namely the chromosomal TbpE and TfbG and the minichromosomal TbpB, were identified by mass spectrometric analysis (Supplemental Table I). Two peptides of TbpE (P = 1) were found for a total of 14 times. The TbpB protein (P = 1), encoded by both pNRC100 and pNRC200, had a peptide identified once. The only detectable Tfb, TfbG (P = 1), had one peptide identified 10 times. The transcription initiation factor IIE α subunit (TfeA) had three peptides identified a total of 17 times. In addition, one peptide each from the termination-antitermination factors NusA and NusG was also identified. Among the 27 transcription regulators predicted from the genome sequence (2Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Crossref PubMed Scopus (600) Google Scholar), 12 were identified in this study (Supplemental Table I). These included the putative regulator ArcR expressed from a gene in the arcRACB gene cluster on pNRC200 that also codes the enzymes for arginine deiminase (ArcA), carbamate kinase (ArcC), and catabolic ornithine transcarbamylase (ArcB), all of which are required for fermentative growth using the arginine deiminase pathway (30Ruepp A. Soppa J. Fermentative arginine degradation in Halobacterium salinarium (formerly Halobacterium halobium): genes, gene products, and transcripts of the arcRACB gene cluster.J. Bacteriol. 1996; 178: 4942-4947Crossref PubMed Google Scholar). The phosphate transport system regulatory protein PhoU and phosphate regulatory protein homolog Prp1 encoded by genes in the phosphate transporter phoU-pstB2A2C2-phoX gene cluster and the juxtaposed downstream gene prp1 were identified. The putative transcription regulators ArsC, CinR, SirR, Trh1, Trh4, Trh5, and Trh7 and Hox-like transcription regulators (Hlx1 and Hlx2) were also detected by mass spectrometry. A total of 117 proteins have been predicted to be associated with the translation in Halobacterium sp. NRC-1 (2Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. Welti R. Goo Y.A. Leithauser B. Keller K. Cruz R. Danson M.J. Hough D.W. Maddocks D.G. Jablonski P.E. Krebs M.P. Angevine C.M. Dale H. Isenbarger T.A. Peck R.F. Pohlschroder M. Spudich J.L. Jung K.W. Alam M. Freitas T. Hou S. Daniels C.J. Dennis P.P. Omer A.D. Ebhardt H. Lowe T.M. Liang P. Riley M. Hood L. DasSarma S. Genome sequence of Halobacterium species NRC-1.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12176-12181Crossref PubMed Scopus (600) Google Scholar). Among these 92 (78.6%) were identified in this study (Supplemental Table I). These included the ribosome structural proteins and proteins involved in aminoacyl-tRNA synthesis, translation initiation, elongation, and release of synthesized polypeptides. Except for the asparaginyl- and glutaminyl-tRNA synthetases, all other synthetase-coding genes have been found in the Halobacterium sp. NRC-1 genome, and these included the duplicated tryptophanyl-tRNA synthetase genes (trpS1 and trpS2) (2Ng W.V. Kennedy S.P. Mahairas G.G. Berquist B. Pan M. Shukla H.D. Lasky S.R. Baliga N.S. Thorsson V. Sbrogna J. Swartzell S. Weir D. Hall J. Dahl T.A. We
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