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 DeinococcusThermus, 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 de- Cyclic AMP (cAMP) receptor proteins (CRPs) are global transcriptional regulators broadly distributed in bacteria (30,72). The cellular roles of such CRP family proteins are diverse and include carbohydrate metabolism (3, 30), development of competence for transformation (8), modulation of virulence gene expression and pathogenesis (10,11,55,57,65), resuscitation (50), and germination and morphological development (13,49).Escherichia coli CRP controls the activity of over 100 genes and has been the most extensively studied so far (30, 72). This CRP was first named the catabolite gene-activating protein, since it induces the transcription of a number of genes in response to carbon source limitation (16,73). In the absence of a carbon source such as glucose, the intracellular cAMP level increases, resulting in the formation of a CRP-cAMP complex, which binds to specific DNA sequences at target promoters. The CRP-cAMP regulatory complex is also involved in the regulation of genes that are not directly related to catabolism (3). In addition, the complex acts as a negative regulator of transcription at cya gene promoter cyaP2, gal operon promoter galP2, crp gene promoter crpP, and deo operon promoter deoP2 (3). E. coli CRP is a dimer of two identical subunits, each 209 residues in length, and contains a helix-turn-helix DNA-binding motif in its C-terminal domain (40). Each subunit can bind one molecule of allosteric effector cAMP. This CRP undergoes a conformational change upon cAMP binding (21,66,67), and the CRP-cAMP complex interacts with a 22-bp DNA site exhibiting twofold symmetry, with the consensus sequence 5Ј-AAATGTGATCTAGATCACATTT-3Ј (15). Biochemical and genetic analyses have revealed that this CRP interacts with the C-terminal domain of the RNA polymerase (RNAP) ␣ subunit (␣CTD) (5,6,24,35,43,44,58). This interaction is thought to facilitate RNAP binding to the promoter, which leads to the formation of an open complex and induction of transcription initiation. Crystallographic studies on E. coli CRP have been performed to determine the structure of CRP-cAMP and the mechanisms underlying the interactions among CRP-cAMP, DNA, and RNAP ␣CTD (30, 34).CRP homologs have been found not only in othe...

Identification of Promoters Recognized by RNA Polymerase-σ E Holoenzyme from Thermus thermophilus HB8

“…2). The E -disruptant (⌬sigE) strain isolated in basically the same way as that described previously (7,23) was not lethal, although the lag phase was longer than that in the case of the wild type, indicating that this gene is not essential for this strain (Fig. 2).…”

“…We observed the expression profile of T. thermophilus sigE mRNA in vivo during cultivation in a rich medium consisting of 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl 2 , and 0.4 mM MgCl 2 , which was adjusted to pH 7.2 with NaOH at 70°C, using GeneChip technology as described previously (23). The mRNA was constitutively expressed under the experimental conditions used, and the level varied ϳ1.5-fold (Fig.…”

“…The three wild-type and the three ⌬sigE strains were cultured for 8 h (A 600 ϭ 3 to 4) in the rich medium. From each strain, crude RNA was extracted, and then cDNA was synthesized from 10 g of the total RNA, followed by fragmentation and labeling with biotin-dideoxy UTP, as described previously (23). The 3Ј-terminal-labeled cDNA (2 g) was hybridized to a TTHB8401 GeneChip (Affymetrix, Santa Clara, CA), and then the array was washed, stained, and scanned as described previously (23).…”

Degradation of ppGpp by Nudix Pyrophosphatase Modulates the Transition of Growth Phase in the Bacterium Thermus thermophilus

“…The RNA isolation and hybridization was performed as described previously (25). Sample preparation was performed four times independently, and the total RNA from each sample was hybridized on a different array.…”

Bacterial Genome Instability

“…Moreover, certain cellular factors and pathways can control the activation of some CRISPR-Cas systems. In Thermus thermophilus, phage infections induce the transcription of cas genes and CRISPR arrays by a sensing mechanism using the cell's cyclic AMP receptor proteins (301,302). Stress, such as phage infection, the accumulation of misfolded proteins in the E. coli membrane, or the absence of ClpP in Streptococcus mutans, can also activate the expression of certain Cas proteins (303,304).…”