Project description:We observed the expression profile of the total mRNA in Thermus thermophilus HB8 wild-type strain at 60 min after the addition of silicic acid and removal of iron.
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.
Project description:We observed the expression profile of the total mRNA in Thermus thermophilus HB8 wild-type strain at 60 min after the addition of silicic acid and removal of iron. Three wild-type strain of T. thermophilus HB8 were pre-cultured at 70 degC for 16 hours in 3 mL of TM medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl and 1% Castenholz' basal salt solution, which was adjusted to pH 7.2 with NaOH. The cells (1 mL) were inoculated into 250 mL of the synthetic medium and then cultivated at 70°A600 value being ~0.60). Then the 600 ppm of silicic acid was added into the medium or removal of iron, and continued cultivation. Cells were collected at 60 min time point after the change of medium, and then crude RNA was extracted. The expression of each mRNA at each time point was analyzed on a GeneChip.
Project description:We observed the expression profile of the total mRNA in Thermus thermophilus HB8 wild-type strain at 0 and 30 min after the addition of ZnSO4.
Project description:We observed the expression profile of the total mRNA of crp (TTHA1437) deletion mutant of Thermus thermophilus HB8 strain grown in a rich (TT) medium at 70 degC
