Dataset Information

13C metabolic flux analysis of three divergent extremely thermophilic bacteria: Geobacillus sp. LC300, Thermus thermophilus HB8, and Rhodothermus marinus DSM 4252.

ABSTRACT: Thermophilic organisms are being increasingly investigated and applied in metabolic engineering and biotechnology. The distinct metabolic and physiological characteristics of thermophiles, including broad substrate range and high uptake rates, coupled with recent advances in genetic tool development, present unique opportunities for strain engineering. However, poor understanding of the cellular physiology and metabolism of thermophiles has limited the application of systems biology and metabolic engineering tools to these organisms. To address this concern, we applied high resolution 13C metabolic flux analysis to quantify fluxes for three divergent extremely thermophilic bacteria from separate phyla: Geobacillus sp. LC300, Thermus thermophilus HB8, and Rhodothermus marinus DSM 4252. We performed 18 parallel labeling experiments, using all singly labeled glucose tracers for each strain, reconstructed and validated metabolic network models, measured biomass composition, and quantified precise metabolic fluxes for each organism. In the process, we resolved many uncertainties regarding gaps in pathway reconstructions and elucidated how these organisms maintain redox balance and generate energy. Overall, we found that the metabolisms of the three thermophiles were highly distinct, suggesting that adaptation to growth at high temperatures did not favor any particular set of metabolic pathways. All three strains relied heavily on glycolysis and TCA cycle to generate key cellular precursors and cofactors. None of the investigated organisms utilized the Entner-Doudoroff pathway and only one strain had an active oxidative pentose phosphate pathway. Taken together, the results from this study provide a solid foundation for future model building and engineering efforts with these and related thermophiles.

SUBMITTER: Cordova LT

PROVIDER: S-EPMC5845442 | biostudies-other | 2017 Nov

REPOSITORIES: biostudies-other

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<sup>13</sup>C metabolic flux analysis of three divergent extremely thermophilic bacteria: Geobacillus sp. LC300, Thermus thermophilus HB8, and Rhodothermus marinus DSM 4252.

Cordova Lauren T LT Cipolla Robert M RM Swarup Adti A Long Christopher P CP Antoniewicz Maciek R MR

Metabolic engineering 20171014

Thermophilic organisms are being increasingly investigated and applied in metabolic engineering and biotechnology. The distinct metabolic and physiological characteristics of thermophiles, including broad substrate range and high uptake rates, coupled with recent advances in genetic tool development, present unique opportunities for strain engineering. However, poor understanding of the cellular physiology and metabolism of thermophiles has limited the application of systems biology and metaboli ...[more]

PMID: 29037779

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Project description:Lysine acetylation in proteins has recently been globally identified in bacteria and eukaryotes. Even though acetylproteins are known to be involved in various cellular processes, its physiological significance has not yet been resolved. Using a proteomics approach in combination with immunoprecipitation, we identified 197 lysine acetylation sites and 4 N-terminal acetylation sites from 128 proteins in Thermus thermophilus HB8, an extremely thermophilic eubacterium. Our analyses revealed that identified acetylproteins are well conserved across all three domains of life and are mainly involved in central metabolism and translation. To further characterize functional significance, we successfully mapped 113 acetylation sites on their 54 authentic and 59 homologous protein structures. The acetylation in the majority of proteins occurs in ordered structures and the sites were situated near the negatively charged glutamic acid residues. In addition, 59 of 103 acetylations were located within considerable distance that can disrupt electrostatic interactions and hydrogen bonding networks on protein surface, demonstrating the physiological significances of the acetylations. Finally, we further summarized 22 critical acetylation sites related to Schiff-base formation, ligand binding, protein-RNA and protein-protein interaction. The structural information of 113 acetylation sites provides new molecular insight into the role of lysine acetylation in the proteins. Data processing, bioinformatics: MS and MS/MS spectral data were processed by DataAnalysis 4.0 software (Bruker Daltonics). The peak lists containing m/z of precursor ions with that of their product ions were generated by the Compound-Auto MS(n) option of the DataAnalysis 4.0 software. Fifty non-deconvoluted peaks over the intensity threshold 150 and charge deconvoluted peaks in each MS/MS spectrum were exported into peak list files. The spectra were searched against our in-house T. thermophilus HB8 database, containing 2,238 protein sequence entries from the complete genome sequence using Mascot search engine (version 2.3; Matrix Science, London, UK). The acetylated peptides were identified using a mass tolerance of ±0.05 Da for precursor and product ions and allowed a maximum of 6 mis-cleavage sites for trypsin. The carbamidomethylation of cysteine was selected as a fixed modification. The oxidation of methionine, deamidation of asparagine and glutamine, acetylation of lysine and acetylation of protein N-terminus were selected as variable modifications. Only peptides in the confidence range of 99% probability (P value < 0.01) in Mascot ion score were assumed to be identified.

Dataset Information

13C metabolic flux analysis of three divergent extremely thermophilic bacteria: Geobacillus sp. LC300, Thermus thermophilus HB8, and Rhodothermus marinus DSM 4252.

Publications

<sup>13</sup>C metabolic flux analysis of three divergent extremely thermophilic bacteria: Geobacillus sp. LC300, Thermus thermophilus HB8, and Rhodothermus marinus DSM 4252.

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