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Genome-scale reconstruction and analysis of the metabolic network in the hyperthermophilic archaeon Sulfolobus solfataricus.

ABSTRACT: We describe the reconstruction of a genome-scale metabolic model of the crenarchaeon Sulfolobus solfataricus, a hyperthermoacidophilic microorganism. It grows in terrestrial volcanic hot springs with growth occurring at pH 2-4 (optimum 3.5) and a temperature of 75-80°C (optimum 80°C). The genome of Sulfolobus solfataricus P2 contains 2,992,245 bp on a single circular chromosome and encodes 2,977 proteins and a number of RNAs. The network comprises 718 metabolic and 58 transport/exchange reactions and 705 unique metabolites, based on the annotated genome and available biochemical data. Using the model in conjunction with constraint-based methods, we simulated the metabolic fluxes induced by different environmental and genetic conditions. The predictions were compared to experimental measurements and phenotypes of S. solfataricus. Furthermore, the performance of the network for 35 different carbon sources known for S. solfataricus from the literature was simulated. Comparing the growth on different carbon sources revealed that glycerol is the carbon source with the highest biomass flux per imported carbon atom (75% higher than glucose). Experimental data was also used to fit the model to phenotypic observations. In addition to the commonly known heterotrophic growth of S. solfataricus, the crenarchaeon is also able to grow autotrophically using the hydroxypropionate-hydroxybutyrate cycle for bicarbonate fixation. We integrated this pathway into our model and compared bicarbonate fixation with growth on glucose as sole carbon source. Finally, we tested the robustness of the metabolism with respect to gene deletions using the method of Minimization of Metabolic Adjustment (MOMA), which predicted that 18% of all possible single gene deletions would be lethal for the organism.

SUBMITTER: Ulas T

PROVIDER: S-EPMC3432047 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Genome-scale reconstruction and analysis of the metabolic network in the hyperthermophilic archaeon Sulfolobus solfataricus.

Ulas Thomas T Riemer S Alexander SA Zaparty Melanie M Siebers Bettina B Schomburg Dietmar D

PloS one 20120831 8

We describe the reconstruction of a genome-scale metabolic model of the crenarchaeon Sulfolobus solfataricus, a hyperthermoacidophilic microorganism. It grows in terrestrial volcanic hot springs with growth occurring at pH 2-4 (optimum 3.5) and a temperature of 75-80°C (optimum 80°C). The genome of Sulfolobus solfataricus P2 contains 2,992,245 bp on a single circular chromosome and encodes 2,977 proteins and a number of RNAs. The network comprises 718 metabolic and 58 transport/exchange reaction ...[more]

PMID: 22952675

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Project description:Sulfolobus solfataricus is an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group of Sulfolobales, N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of the N-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood in Archaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening of S. solfataricus extracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified in Archaea Mass spectrometry analysis of the purified activity showed a protein encoded by the sso2901 gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed in S. solfataricus extracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream of sso2901 (sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism in S. solfataricusIMPORTANCE In this study, a preliminary screening of cellular extracts of S. solfataricus allowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism of Archaea.

Project description:BackgroundReverse gyrases are DNA topoisomerases characterized by their unique DNA positive-supercoiling activity. Sulfolobus solfataricus, like most Crenarchaeota, contains two genes each encoding a reverse gyrase. We showed previously that the two genes are differently regulated according to temperature and that the corresponding purified recombinant reverse gyrases have different enzymatic characteristics. These observations suggest a specialization of functions of the two reverse gyrases. As no mutants of the TopR genes could be obtained in Sulfolobales, we used immunodetection techniques to study the function(s) of these proteins in S. solfataricus in vivo. In particular, we investigated whether one or both reverse gyrases are required for the hyperthermophilic lifestyle.ResultsFor the first time the two reverse gyrases of S. solfataricus have been discriminated at the protein level and their respective amounts have been determined in vivo. Actively dividing S. solfataricus cells contain only small amounts of both reverse gyrases, approximately 50 TopR1 and 125 TopR2 molecules per cell at 80°C. S. solfataricus cells are resistant at 45°C for several weeks, but there is neither cell division nor replication initiation; these processes are fully restored upon a return to 80°C. TopR1 is not found after three weeks at 45°C whereas the amount of TopR2 remains constant. Enzymatic assays in vitro indicate that TopR1 is not active at 45°C but that TopR2 exhibits highly positive DNA supercoiling activity at 45°C.ConclusionsThe two reverse gyrases of S. solfataricus are differently regulated, in terms of protein abundance, in vivo at 80°C and 45°C. TopR2 is present both at high and low temperatures and is therefore presumably required whether cells are dividing or not. By contrast, TopR1 is present only at high temperature where the cell division occurs, suggesting that TopR1 is required for controlling DNA topology associated with cell division activity and/or life at high temperature. Our findings in vitro that TopR1 is able to positively supercoil DNA only at high temperature, and TopR2 is active at both temperatures are consistent with them having different functions within the cells.

Dataset Information

Genome-scale reconstruction and analysis of the metabolic network in the hyperthermophilic archaeon Sulfolobus solfataricus.

Publications

Genome-scale reconstruction and analysis of the metabolic network in the hyperthermophilic archaeon Sulfolobus solfataricus.

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