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Transcription activation mediated by a cyclic AMP receptor protein from Thermus thermophilus HB8.

ABSTRACT: The extremely thermophilic bacterium Thermus thermophilus HB8, which belongs to the phylum Deinococcus-Thermus, has an open reading frame encoding a protein belonging to the cyclic AMP (cAMP) receptor protein (CRP) family present in many bacteria. The protein named T. thermophilus CRP is highly homologous to the CRP family proteins from the phyla Firmicutes, Actinobacteria, and Cyanobacteria, and it forms a homodimer and interacts with cAMP. CRP mRNA and intracellular cAMP were detected in this strain, which did not drastically fluctuate during cultivation in a rich medium. The expression of several genes was altered upon disruption of the T. thermophilus CRP gene. We found six CRP-cAMP-dependent promoters in in vitro transcription assays involving DNA fragments containing the upstream regions of the genes exhibiting decreased expression in the CRP disruptant, indicating that the CRP is a transcriptional activator. The consensus T. thermophilus CRP-binding site predicted upon nucleotide sequence alignment is 5'-(C/T)NNG(G/T)(G/T)C(A/C)N(A/T)NNTCACAN(G/C)(G/C)-3'. This sequence is unique compared with the known consensus binding sequences of CRP family proteins. A putative -10 hexamer sequence resides at 18 to 19 bp downstream of the predicted T. thermophilus CRP-binding site. The CRP-regulated genes found in this study comprise clustered regularly interspaced short palindromic repeat (CRISPR)-associated (cas) ones, and the genes of a putative transcriptional regulator, a protein containing the exonuclease III-like domain of DNA polymerase, a GCN5-related acetyltransferase homolog, and T. thermophilus-specific proteins of unknown function. These results suggest a role for cAMP signal transduction in T. thermophilus and imply the T. thermophilus CRP is a cAMP-responsive regulator.

SUBMITTER: Shinkai A

PROVIDER: S-EPMC1913326 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Transcription activation mediated by a cyclic AMP receptor protein from Thermus thermophilus HB8.

Shinkai Akeo A Kira Satoshi S Nakagawa Noriko N Kashihara Aiko A Kuramitsu Seiki S Yokoyama Shigeyuki S

Journal of bacteriology 20070316 10

The extremely thermophilic bacterium Thermus thermophilus HB8, which belongs to the phylum Deinococcus-Thermus, has an open reading frame encoding a protein belonging to the cyclic AMP (cAMP) receptor protein (CRP) family present in many bacteria. The protein named T. thermophilus CRP is highly homologous to the CRP family proteins from the phyla Firmicutes, Actinobacteria, and Cyanobacteria, and it forms a homodimer and interacts with cAMP. CRP mRNA and intracellular cAMP were detected in this ...[more]

PMID: 17369302

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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

Transcription activation mediated by a cyclic AMP receptor protein from Thermus thermophilus HB8.

Publications

Transcription activation mediated by a cyclic AMP receptor protein from Thermus thermophilus HB8.

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