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Resolution of Carbon Metabolism and Sulfur-Oxidation Pathways of Metallosphaera Cuprina Ar-4 Via Comparative Proteomics

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Resolution of Carbon Metabolism and Sulfur-Oxidation Pathways of Metallosphaera Cuprina Ar-4 Via Comparative Proteomics JOURNAL OF PROTEOMICS 109 (2014) 276– 289 Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jprot Resolution of carbon metabolism and sulfur-oxidation pathways of Metallosphaera cuprina Ar-4 via comparative proteomics Cheng-Ying Jianga, Li-Jun Liua, Xu Guoa, Xiao-Yan Youa, Shuang-Jiang Liua,c,⁎, Ansgar Poetschb,⁎⁎ aState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China bPlant Biochemistry, Ruhr University Bochum, Bochum, Germany cEnvrionmental Microbiology and Biotechnology Research Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China ARTICLE INFO ABSTRACT Article history: Metallosphaera cuprina is able to grow either heterotrophically on organics or autotrophically Received 16 March 2014 on CO2 with reduced sulfur compounds as electron donor. These traits endowed the species Accepted 6 July 2014 desirable for application in biomining. In order to obtain a global overview of physiological Available online 14 July 2014 adaptations on the proteome level, proteomes of cytoplasmic and membrane fractions from cells grown autotrophically on CO2 plus sulfur or heterotrophically on yeast extract Keywords: were compared. 169 proteins were found to change their abundance depending on growth Quantitative proteomics condition. The proteins with increased abundance under autotrophic growth displayed Bioleaching candidate enzymes/proteins of M. cuprina for fixing CO2 through the previously identified Autotrophy 3-hydroxypropionate/4-hydroxybutyrate cycle and for oxidizing elemental sulfur as energy Heterotrophy source. The main enzymes/proteins involved in semi- and non-phosphorylating Entner– Industrial microbiology Doudoroff (ED) pathway and TCA cycle were less abundant under autotrophic growth. Also Extremophile some transporter proteins and proteins of amino acid metabolism changed their abundances, suggesting pivotal roles for growth under the respective conditions. Abbreviations: ED pathway, Entner–Doudoroff pathway; EMP, Embden–Meyerhof–Parnas pathway; GA, glyceraldehyde; GAP, glyceralde- hyde 3-phosphate; KDG, 2-keto-3-deoxygluconate; KD(P)G, 2-keto-3-deoxy-(6-phospho)-gluconate; PEP, phosphoenolpyruvate; GDH, glucose dehydrogenase; GAD, gluconate dehydratase; GAOR, glyceraldehyde oxidoreductase; GADH, glyceraldehyde dehydratase; GAPDH, phosphorylating GAP dehydrogenase; GAPN, non-phosphorylating GAP, dehydrogenase; GAPOR, GAP oxidoreductase; PGK, phosphoglyc- erate kinase; PGM, phosphoglycerate mutase; FBPA/ase, fructose 1,6-bisphosphate aldolase/bisphosphatase; FBPA, fructose 1,6-bisphosphate aldolase; TIM, triosephosphate isomerase; BPG, 2,3-bisphosphoglycerate; PGI/PMI, bifunctional phosphoglucose/phosphomannose isomer- ase; PGM/PMM, phosphoglucomutase/phosphomannomutase; 3-HP/4-HB, 3-hydroxypropionyl/4-hydroxybutyryl; ACC/PCC, acetyl-CoA carboxylase/propionyl-CoA carboxylase; MSR, malonate semialdehyde reductase; 4-HBCS, 4-hydroxybutyryl-CoA synthetase; 4-HBCD, 4-hydroxybutyryl-CoA dehydratase; SSR, succinic semialdehyde reductase; ACK, acetoacetyl-CoA β-ketothiolase; 3-HPCD, 3-hydroxypropionyl- CoA dehydratase; ACR, acryloyl-CoA reductase; MSR, malonate semialdehyde reductase; 3-HPCS, 3-hydroxypropionyl-CoA synthetase (AMP-forming); MCE, methylmalonyl-CoA epimerase; MCM, methylmalonyl-CoA mutase; CCH, crotonyl-CoA hydratase; SOR, sulfur oxidoreductase; SQR, sulfide quinone oxidoreductase; NSR, NADPH:sulfur oxidoreductase; SR, sulfite reductase; SAOR, sulfite:acceptor oxidoreductase; TQO, thiosulfate:quinone oxidoreductase; TetH, tetrathionate hydrolase; Hdr, heterodisulfide reductase; APS, adenosine-5′- phosphosulfate; APSR, adenosine-5′-phosphosulfate reductase; APAT, adenylylsulfate:phosphate adenylyltransferase; AK, adenylate kinase. ⁎ Correspondence to: S.-J. Liu, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China. Tel.: +86 10 64847423; fax: +86 10 64807421. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (S.-J. Liu), [email protected] (A. Poetsch). http://dx.doi.org/10.1016/j.jprot.2014.07.004 1874-3919/© 2014 Elsevier B.V. All rights reserved. JOURNAL OF PROTEOMICS 109 (2014) 276– 289 277 Biological significance The described work is of great significance: For general microbiology: • How do extremophile organisms use their unique metabolic capabilities in adapting to autotrophic and hetetrotrophic growth conditions? • Which are important enzymes involved in the metabolic adaptation and which enzyme candidateshouldbeinvestigatedinmoredetailwith microbiological/biochemical approaches? For applied microbiology: • Which are the key enzymes and reaction pathways for sulfur oxidation and autotrophic growth? This knowledge should accelerate future design of improved bioleaching processes in biomining industries or bioremediation. © 2014 Elsevier B.V. All rights reserved. 1. Introduction at an optimal temperature of 65 °C and pH of 3.5. Strain Ar-4 grows chemolithoautotrophically on CO2 by oxidation of Extremely thermoacidophilic Archaea from the order elemental sulfur, ferrous iron, tetrathionate and pyrite, it Sulfolobales, including genus Sulfolobus, Acidianus,and also grows chemoorganoheterotrophically on various organic Metallosphaera, contribute greatly to biogeochemical element compounds such as yeast extract, amino acids, and in con- cycling and biohydrometallurgical processes with their physio- trast to M. sedula, D-glucose and few other monosaccharides logical versatility. So far, the most extensively studied model [8]. The M. cuprina genome is 16% smaller than the M. sedula organism for Crenarchaeotes has been Sulfolobus solfataricus. The genome, however, it harbors 233 ORFs that do not occur in complete arsenal of systems biology (genomics, transcriptomics, M. sedula[10]. – proteomics, metabolomics) together with modeling tools had Since 2D E is limited in its proteome coverage (membrane been used to understand its physiology under various condi- & alkalic proteins), we selected quantitative shotgun proteo- tions [1].However,Sulfolobus spp. lack or easily lost many mics instead to obtain a comprehensive and comparative physiological features, such as the iron or sulfur-oxidizing view on the physiology of M. cuprina under autotrophic and ability, exploited in biomining industries under autotrophic organotrophic growth. This first large-scale proteomics study growth condition [2]. Metallosphaera spp. stand out from for the genus Metallosphaera enabled a detailed description of Sulfolobales by their ability to grow heterotrophically on differences in central carbon metabolism, enzymes of the 3-HP/4-HB cycle, and oxidation pathway of RISCs. organics, and autotrophically by fixing CO2 with hydrogen, ferrous or reduced inorganic sulfur compounds (RISCs) as electronic donor or directly mobilizing metals from metal sulfides. The availability of the Metallosphaera sedula genome 2. Materials and methods sequence has enabled -omics studies, which mainly aimed M. cuprina at further elucidation of the CO2 fixation pathway and the 2.1. Cultivation of Ar-4 electron transport chain. DNA microarray analysis of M. sedula grown on yeast extract supplemented with different sulfur M. cuprina Ar-4 was cultivated aerobically at 65 °C in base salt compounds was used to identify the enzymes involved in medium (BSM) [10], supplemented with 5 g/L elemental sulfur iron and sulfur oxidation [3]. In addition autotrophic, hetero- (for autotrophic growth) or 2 g/L yeast extract (for heterotrophic trophic and mixotrophic growths were characterized [4] to growth). Autotrophic cultures were shaked three times every day narrow-down enzyme candidates for the 3-hydroxypropionate/ to maintain micro amount of CO2 in medium. Heterotrophic 4-hydroxybutyrate (3-HP/4-HB) cycle. Cultivation under strict culture was carried out without agitation. For proteome analysis, – H2 CO2 autotrophy was realized by growing M. sedula in a four 1-L flasks with aseptic filtration membrane, each containing bioreactor and transcriptomics together with biochemical 300-mL of BSM medium, were used. Cells were continuously assays was used to conclude on the physiologically relevant cultivated either autotrophically or heterotrophically for at least 4-hydroxybutyrate-CoA (4-HB-CoA) synthetase [5].Theobserved three transfers/generations in 100 mL (250 mL flask) before ino- negative impact of hydrogen on bioleaching efficiency of M. culated into 1-L flask. Autotrophically or heterotrophically grown sedula was confirmed at molecular level with transcriptomics [6]. cells were harvested in the late exponential growth phase (with 7– 7 – Metabolic flux analyses revealed interesting facts about carbon cell densities of 4 × 10 6×10/mL (OD600 =0.06 0.08) for auto- 8– 8 – metabolism, notably succinyl-CoA and not acetyl-CoA as main trophic cells and 2 × 10 4×10/mL (OD600 =0.3 0.5)), Fig. S1. anabolic precursor [7]. So far only once, proteomics, a compar- ison of M. sedula strains with high and low copper tolerance 2.2. Preparation of proteome fractions (2D-electrophoresis), was employed [8]. Metallosphaera cuprina Ar-4 was isolated from the muddy Preparation of cytoplasmic and membrane proteomes was water samples of a sulfuric hot spring [9]. It grows aerobically performed according to Haussmann et al. [11].Briefly,M. cuprina 278 JOURNAL OF PROTEOMICS 109 (2014) 276– 289 Ar-4 cells were harvested by centrifugation for 15 min at 8000 ×g. loaded onto a triphasic microcapillary column as described Cells were washed twice with
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