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.

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 5′-AAATGTGATCTAGATCACATTT-3′ (15). Biochemical and genetic analyses have revealed that this CRP interacts with the C-terminal domain of the RNA polymerase (RNAP) α subunit (αCTD) (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 αCTD (30, 34).

CRP homologs have been found not only in other members of the Enterobacteriaceae family, such as Salmonella enterica serovar Typhimurium and Shigella flexneri (9), but also in numerous physiologically and ecologically distinct species, such as Haemophilus influenzae (8), Xanthomonas campestris pv. (11), Vibrio cholerae (55), Pseudomonas aeruginosa (57), Synechocystis sp. strain PCC6803 (70), Corynebacterium glutamicum (29), Streptomyces coelicolor (12), and Mycobacterium tuberculosis (50). The consensus binding sequences of CRP homologs, such as C. glutamicum GlxR (36), M. tuberculosis Rv3676 (CRP_Mt) (1), H. influenzae CRP (7), and P. aeruginosa Vfr (28), are similar to that of E. coli CRP.

Thermus thermophilus HB8, which belongs to the phylum Deinococcus-Thermus, is an extremely thermophilic bacterium isolated from the water at a Japanese hot spring that can grow at 47 to 85°C, with its optimum temperature range being 65 to 72°C (46). The genome of T. thermophilus HB8, with a GC content of 69.5%, is composed of 1.85-Mbp chromosomal DNA, 0.26-Mbp plasmid pTT27, and 9.32-kbp plasmid pTT8, which encode 1,973, 251, and 14 open reading frames (ORFs), respectively. With its relatively small genome, this strain is considered one of the minimum models of life. The complete genomic sequence of this strain has been determined (NCBI accession numbers NC_006461, NC_006462, and NC_006463), and structural and functional genomic studies on it have begun (68, 69). T. thermophilus HB8 has a typical bacterial-type RNAP, and its crystal structure has been determined at 2.6-Å resolution (60). The structure provided information about the structural organization of complexes of transcription intermediates as well as the mechanism underlying transcription initiation. Therefore, this strain is an appropriate model for understanding bacterial transcription, including the mechanism underlying transcriptional regulation by transcription factors, at the atomic level. In the T. thermophilus genome, many ORFs encoding probable transcriptional factors have been found; however, their functions, activities, and binding sites remain to be elucidated.

In the T. thermophilus genome, four ORFs, TTHA1437, TTHA1567, TTHA1359, and TTHB099, which exhibit amino acid sequence homology to CRP/FNR (fumarate-nitrate reduction regulator) family transcriptional regulators, have been found (NCBI accession numbers YP_144703, YP_144833, YP_144625, and YP_145338). In this study, we performed functional analysis of one of the CRP homologs, TTHA1437 protein, and showed that it acts as a transcriptional activator of several genes in a cAMP-dependent manner.

MATERIALS AND METHODS

Cloning of CRP gene.

The T. thermophilus CRP gene was amplified by genomic PCR, using 5′-ATATcatatgAAAGGAAGCCCCCTGTTCCACGGCCT-3′ (NdeI site in lowercase) and 5′-ATATagatctTTATTAGGCAAGCCCGAAGGCGATCTCCTCA-3′ (BglII site in lowercase) as primers, and then the amplified fragment was cloned into a TA cloning vector, pT7Blue (Novagen, Madison WI). A 650-bp NdeI-BglII fragment of the plasmid, encoding the CRP gene, was cloned under the control of the T7 promoter (NdeI-BamHI site) of E. coli expression vector pET-11a (Novagen) to construct pET-ttCRP.

Expression and purification of recombinant CRP protein.

E. coli BL21(DE3) (Novagen), containing plasmid pET-ttCRP, was cultured at 37°C in 4 liters of LB broth supplemented with 50 μg/ml ampicillin. When the cell density reached 2 × 10⁸ cells/ml, isopropyl 1-thio-β-d-galactopyranoside (IPTG) was added to a final concentration of 1 mM, and the culture was continued at 37°C for a further 4.5 h. The cells were collected by centrifugation, washed with 40 ml of buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl), and then resuspended in 140 ml of the same buffer. The cells were disrupted by sonication in ice water, and then the cell lysate was incubated at 70°C for 10 min. The sample was centrifuged at 150,000 × g for 1 h at 4°C, and then ammonium sulfate was added to the supernatant to a final concentration of 2 M. The precipitate was collected by centrifugation, suspended in 15 ml of 20 mM Tris-HCl, pH 8.0, and then desalted by fractionation on a HiPrep 26/10 desalting column (GE Healthcare Bio-Science Corp., Piscataway, NJ) preequilibrated with the same buffer. Then the sample was applied to a Resource Q column (GE Healthcare Bio-Science Corp.) preequilibrated with 20 mM Tris-HCl, pH 8.0. The column was washed with the same buffer, and then the bound protein was eluted with a linear gradient of 0 to 1 M NaCl in 20 mM Tris-HCl, pH 8.0. The fractions containing the recombinant T. thermophilus CRP were pooled, desalted on a column as described above, and then applied to a BioScale CHT10-I (Bio-Rad Laboratories, Inc., Hercules, CA) column preequilibrated with 10 mM sodium phosphate, pH 7.0, containing 0.15 M NaCl. The flowthrough fraction was collected, concentrated with a Vivaspin 20 concentrator (5-kDa molecular-mass cutoff; Sartorius AG, Goettingen, Germany), and then applied to a HiLoad 16/60 Superdex 75 (GE Healthcare Bio-Science Corp.) column preequilibrated with 20 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl. The fractions containing the CRP were collected and concentrated with a Vivaspin 6 concentrator (5-kDa molecular-mass cutoff; Sartorius AG). The protein concentration was determined by measuring the absorbance at 280 nm (32).

Proteolytic digestion of T. thermophilus CRP.

The reaction mixture (25 μl), comprising 10 μg of CRP, 20 mM Tris-HCl, pH 8.0, and 150 mM NaCl, with or without 1 mM cAMP, was preincubated at 37°C for 10 min, and then 25 μg (2.5 μl) of Staphylococcus aureus protease V8 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added and the incubation was continued. Aliquots (5 μl) were withdrawn at 15, 30, 45, and 60 min, and the proteolytic digestion was terminated on ice by the addition of 5 μl of a solution comprising 10 mM phenylmethylsulfonyl fluoride, 125 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2-mercaptoethanol, and 0.02% bromophenol blue. All samples were boiled for 5 min and then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE).

Disruption of the T. thermophilus CRP gene.

The plasmid used for the disruption carried the upstream region of the target gene, followed by the thermostable kanamycin-resistant marker (HTK) gene and the downstream region of the target gene, as described previously (20). The 500-bp fragment containing the upstream or downstream region of the CRP gene was amplified by genomic PCR, using 5′-GCCCGGGCCACGGCCACC-3′ and 5′-ATATtctagaGAACAGGGGGCTTCCCTT-3′ (XbaI site in lowercase) as primers for the upstream region and 5′-ATATctgcagATCGCCTTCGGGCTTGCC-3′ (PstI site in lowercase) and 5′-CCCAGGTACTCGGCCAGG-3′ as primers for the downstream region. Each amplified fragment was treated with XbaI and PstI, respectively, to obtain fragments 1 and 2. The HTK gene was amplified by PCR from a plasmid carrying the HTK gene (20), using 5′-ATATtctagaCGTTGACGGCGGGATATG-3′ and 5′-ATATctgcagCGTAACCAACATGATTAA-3′ as primers, and then the amplified fragments were treated with XbaI and PstI to yield fragment 3. Fragments 1, 2, and 3 were ligated into the pGEM-T Easy vector (Promega, Madison, WI) to construct pGEM-ΔCRP. The plasmid was transformed into the T. thermophilus HB8 strain, and three kanamycin-resistant clones were isolated as disruptants as to the CRP gene, as described previously (20). We confirmed that the CRP gene was replaced by the HTK gene by genomic PCR.

DNA microarrays. (i) Cell culture and RNA isolation.

The T. thermophilus wild-type and ΔCRP strains were each precultured at 70°C for 16 h in 3 ml of TT medium comprising 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl₂, and 0.4 mM MgCl₂, which was adjusted to pH 7.2 with NaOH. The cells (2 ml) were inoculated into 1 liter of the same medium and then cultivated at 70°C. Cells were collected from 5 to 200 ml of the culture medium, and then crude RNA was extracted by the addition of 1.4 ml of a solution comprising 5 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.25% SDS, and 50% of water-saturated phenol. This mixture was incubated at 65°C for 5 min, chilled on ice for 5 min, and then centrifuged at 4°C. Then, 750 μl of TRIZOL LS (Invitrogen, Carlsbad, CA) was added to 0.2 ml of the aqueous phase. After incubation for 5 min at room temperature, the RNA was extracted with 0.2 ml of chloroform. The extraction was repeated with 0.5 ml of chloroform, and the aqueous phase was precipitated with isopropanol. The pellet was dissolved in 0.2 ml of nuclease-free water, precipitated with ethanol, and then resuspended in 40 μl of water. The RNA was treated with DNase I (Ambion, Austin, TX) at 37°C for 20 min in a 25-μl reaction mixture. The reaction was terminated by the addition of 1 μl of 0.5 M EDTA, followed by incubation at 70°C for 5 min. Thereafter, the RNA was precipitated with ethanol in the presence of 1% glycogen and 1 M NH₄OAc.

(ii) cDNA synthesis, fragmentation, and labeling.

cDNA was synthesized with SuperScript II (Invitrogen) reverse transcriptase in the presence of RNase inhibitor SUPERase (Ambion) and 6-base random primers (Invitrogen). The cDNA was fragmented with 35 units of DNase I (GE Healthcare Bio-Science Corp.) at 37°C for 10 min, and after inactivation at 98°C for 10 min, the cDNA fragments were labeled with biotin-ddUTP, using terminal transferase according to the manufacturer's instructions (ENZO Biochem. Inc., Farmingdale, NY).

(iii) Gene chip hybridization.

3′-Terminally labeled cDNA (2 μg) was hybridized to a TTHB8401 GeneChip (Affymetrix, Santa Clara, CA), which contained probe sets of 25-mer oligonucleotides representing 2,238 ORFs and 1,096 intergenic regions. The array was incubated for 16 h at 50°C in a solution comprising 180 mM morpholineethanesulfonic acid (MES), pH 6.6, 40 mM EDTA, 0.02% Tween 20, 7% dimethyl sulfoxide, 1 mg of herring sperm DNA (Promega), 0.5 mg of bovine serum albumin (BSA), the recommended amount of eukaryotic hybridization control (Affymetrix), and control oligonucleotide B2 for the alignment signal (Affymetrix), and then the array was automatically washed and stained with streptavidin-phycoerythrin (Invitrogen) by using a GeneChip fluidics station, 450XP (Affymetrix). The probe array was scanned with a GeneArray scanner (Affymetrix).

(iv) Data analysis.

For the time course experiment, the image data for each time point for the three wild-type strains were scaled to 2,238 ORF target intensity by means of the one-step Tukey's biweight algorithm (23) using GeneChip Operating software, version 1.0 (Affymetrix, Santa Clara, CA). Then, the three data for each time point were normalized through the following three normalization steps in the GeneSpring 7.2 program, that is, data transformation (set measurements of less than 0.01 to 0.01), followed by per chip normalization (normalize as to median) and per gene normalization (normalize as to median) (Silicon Genetics, Redwood City, CA).

For determination of the mRNA expression level in the ΔCRP strain relative to that in the wild type, the image data for the three wild-type and three ΔCRP strains were processed by the same procedure as described above except for the per chip normalization, which was performed by using the wild-type data as the control sample (Silicon Genetics). The q value of the observed differences in the normalized intensities between the wild-type and ΔCRP strains was calculated using the R program (http://cran.us.r-project.org/).

Measurement of the intracellular cAMP level.

The T. thermophilus wild-type strain was precultured at 70°C for 16 h in 3 ml of TT medium. The cells (0.2 ml, A₆₀₀ = 8 to 10) were inoculated into 100 ml of the same medium and then cultivated at 70°C. The cells from a 5-ml aliquot of the culture were collected by centrifugation at 8,000 × g for 1 min at 4°C, rapidly washed with 1 ml of ice-cold buffer (50 mM Tris-HCl, pH 7.7, 150 mM NaCl, and 5% glycerol), resuspended in 0.5 ml of ice-cold EIA buffer provided with a cAMP enzyme immunoassay kit (Cayman Chemical Company, Ann Arbor, MI), and then disrupted by sonication in ice water for 1 min, with output and duty parameters of 2 and 50, respectively (UD-201; TOMY, Japan). The protein concentrations of the samples were determined by the Bradford method with BSA as a standard. The samples were boiled for 15 min, and then the denatured proteins were removed by centrifugation. The cAMP concentrations were measured after acetylation of the samples according to the manufacturer's instructions (Cayman Chemical Company). The bacterial cultivation was performed in duplicate, the cAMP measurement was performed in triplicate for each time point, and the average intracellular cAMP level with the standard deviation (SD) value was expressed as pmol per mg of total protein.

In vitro transcription assays. (i) Preparation of template.

The upstream regions of the TTHB186, TTHB178, TTHB147, and TTHA0771 genes were amplified by genomic PCR, using 5′-CTACGCggatccCCTCGGAGTAGGAGCGGGTGGCGT-3′ (BamHI site in lowercase) and 5′-GCTCTTgaattcTTGTCCCATGTGTTCATGCTAAGC-3′ (EcoRI site in lowercase), 5′-ATATAggatccAGCCGGGTGCAAAGGTCACATCCCCCCGTCCGGGACCCTCC-3′ and 5′-ATATgaattcCACGGCCACGGCTTCAAGATAGGAGGGTCCCGGACGGGGGG-3′, 5′-ATATAggatccCCCCGCGTGCACACTTCACACGGGTCCTGGGGAGGCCCGTT-3′ and 5′-ATATgaattcCCCAAGCATGGCCCGAGTATAAACGGGCCTCCCCAGGACCC-3′, and 5′-ATATAggatccGGGTTCGGTCATTTTTCACAGGGGTTTGACAGACGAGCGT-3′ and 5′-ATATgaattcGCTCTCTCGCCATGCAGTTTAACGCTCGTCTGTCAAACCCC-3′ as primers, respectively. The amplified fragments were digested with BamHI or EcoRI and then cloned into pUC19.

For the construction of plasmids containing the upstream regions of TTHA0176 and THB159, oligonucleotides 5′-ATATAggatccGGTCCAGGGCCTTTTTCACATGCCTCTCAGGGGGTGCGGGG-3′ and 5′-ATATgaattcACGTGCATCGCCCCCATGCTACCCCGCACCCCCTGAGAGGC-3′, and 5′-ATATAggatccGGGCCCGTGCAGAGATCACAACGCCTCGGCCCTGGCGGGAG-3′ and 5′-ATATgaattcAACATAGCGATCTCCCATTTTCTCCCGCCAGGGCCGAGGC-3′, respectively, were annealed, and the partially duplex oligonucleotides were extended by incubation with E. coli DNA polymerase I Klenow fragment (New England BioLabs, Beverly, MA) as described previously (54). The resulting DNA fragments were digested with BamHI or EcoRI, purified on a 0.8% agarose gel, and then cloned into pUC19.

Using each plasmid as a template, PCR was performed to prepare template DNA for the transcription assay using 5′-TGCATGCCTGCAGGTCGACT-3′ and 5′-GATCGGTGCGGGCCTCTTCG-3′ as primers. Various lengths of template DNA containing the upstream region of the TTHB186 gene were prepared using 5′-GATCGGTGCGGGCCTCTTCG-3′ and 5′-GGGCGCTGGTCCCAGTATAG-3′ (for template a), 5′-TAGCATGGTCAAAAATCACA-3′ (for template b), 5′-AATCACACCCTGGCCCAAAC-3′ (for template c), and 5′-TGGCCCAAACGGCTTGCTTA-3′ (for template d) as primers. The amplified fragments were excised from a 0.8% agarose gel, extracted with phenol and ether, and then precipitated with ethanol. The DNA fragments were used for the following assay.

(ii) Runoff transcription.

Assays were performed in 15-μl reaction mixtures in the presence of 1 μM T. thermophilus CRP, 1 μM CRP, and 2 mM cAMP, or in the absence of both. The buffer conditions were based on those reported previously (64). The reaction mixture was composed of buffer A (50 mM glycine-NaOH, pH 8.5, 0.1 mM EDTA, 5 mM dithiothreitol, 18 mM MgCl₂, and 50 mM KCl), 0.3 mM concentrations of each recombinant nucleoside triphosphate (rNTP), 1.5 μCi [α-³²P]CTP (GE Healthcare Bio-Science Corp.), 200 nM template DNA, 50 nM RNAP holoenzyme from T. thermophilus HB8 (T. thermophilus RNAP) purified as described previously (61), and 50 μg/ml BSA. The template DNA was preincubated with or without the CRP, in the presence or absence of 2 mM cAMP, at 55°C for 5 min. RNAP was then added, and the mixture was incubated for a further 5 min. Transcription was initiated by the addition of 1.5 μCi [α-³²P]CTP (GE Healthcare Bio-Science Corp.) and unlabeled rNTPs. After a further 10 min of incubation, the reaction was stopped by the addition of 11 μl of a stop mix composed of 20 mM EDTA, 96% (vol/vol) deionized formamide, 0.01% bromophenol blue, and 0.01% xylene cyanol. The sample was analyzed on a 10% polyacrylamide gel containing 8 M urea, with visualization by autoradiography.

Identification of transcription start site.

RNA was synthesized at 55°C for 20 min, in a 100-μl reaction mixture composed of buffer A, 200 nM template, 100 nM T. thermophilus RNAP, 0.3 mM concentrations of each rNTP, 50 μg/ml BSA, 1 μM T. thermophilus CRP, and 2 mM cAMP. The sample was treated with 5 units of RNase-free DNase I (Takara Bio Inc., Shiga, Japan) at 37°C for 10 min. After the DNase had been inactivated by heat treatment followed by phenol extraction, the RNA was precipitated with 2-propanol in the presence of 1% glycogen. The sample was dissolved in 100 μl H₂O and then precipitated with ethanol. Using the RNA as a template and 5′-GATCGGTGCGGGCCTCTTCG-3′ as a primer, cDNA was synthesized with 2.5 units of avian myeloblastosis virus reverse transcriptase XL (Takara Bio Inc.) in a 20-μl reaction mixture, containing 0.5 mM each dNTP and 25 μCi [α-³²P]dCTP (GE Healthcare Bio-Science Corp.), at 42°C for 20 min. The reaction was stopped by the addition of 15 μl of the stop mix. The nucleotide sequence of the template DNA was determined by the dideoxy-mediated chain termination method (51), using T7 DNA polymerase (GE Healthcare Bio-Science Corp.). Samples were analyzed on an 8% polyacrylamide gel containing 8 M urea, with visualization by autoradiography.

Other methods.

N-terminal sequence analysis was performed with a protein sequencer (Procise HT; Applied Biosystems, Foster City, CA). SDS-PAGE was performed with precast 5 to 20% (wt/wt) polyacrylamide gels (ATTO Corp., Tokyo, Japan), according to the method of Laemmli (33), and the gels were stained with Coomassie brilliant blue R-250. Gel filtration chromatography to estimate the molecular mass of T. thermophilus CRP was performed with a HiLoad 16/60 Superdex 75 column. Dynamic light scattering photometry was performed with a DynaPro-801 detector (Protein Solutions Inc., Charlottesville, VA). Blast and conserved domain database searches were performed on websites http://www.ncbi.nlm.nih.gov/BLAST/ and http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, respectively.

The microarray data discussed in this study have been deposited in the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE7175.

RESULTS

Amino acid sequence of T. thermophilus CRP.

The TTHA1437 ORF from T. thermophilus HB8 encodes 216 amino acid residues (NCBI accession number YP_144703) with a predicted molecular mass of 23,818 Da. In the protein database, the translated amino acid sequence of the ORF was used to search for similar and matching amino acid sequences. A BLAST search revealed that the TT_C1072 protein from T. thermophilus HB27 showed the highest e value, 1e-93, with a single amino acid substitution (E189A). Homologous proteins that showed high e values are from Halothermothrix orenii H 168 (3e-30) of the phylum Firmicutes; Anabaena variabilis ATCC 29413 (1e-25), Nostoc sp. strain PCC 7120 (1e-25), Nostoc punctiforme PCC 73102 (4e-24), and Nodularia spumigena CCY9414 (9e-24) of the phylum Cyanobacteria; and Nocardioides sp. strain JS614 (1e-25), Arthrobacter sp. strain FB24 (1e-24), Kineococcus radiotolerans SRS30216 (1e-24), Arthrobacter aurescens TC1 (2e-24), and Brevibacterium linens BL2 (3e-23) of the phylum Actinobacteria (Fig. 1). Among the proteins with known functions, S. coelicolor CRP (12), C. glutamicum GlxR (29), and M. tuberculosis Rv3676 (CRP_Mt) (50) show high e values, i.e., 3e-23, 2e-18, and 1e-17, respectively (Fig. 1). Synechocystis sp. strain PCC6803 belongs to the Cyanobacteria; however, the e value of one of the well-characterized CRP homologs, SYCRP1, from the strain is low (2e-14) (Fig. 1). The phylum γ-Proteobacteria, including the Enterobacteriaceae, Pseudomonadaceae, Xanthomonadaceae, Vibrionaceae, and Pasteurellaceae families, is phylogenetically distant from T. thermophilus HB8, and E. coli CRP, a prototype of the CRP family proteins, exhibited an e value of 4e-14 (Fig. 1).

FIG. 1.

Amino acid sequence alignment of T. thermophilus CRP with representative homologous proteins. The amino acid sequences of T. thermophilus CRP (TT), H. orenii H 168 HoreDRAFT_0288 (HO), A. variabilis ATCC 29413 Ava_3066 (AV), Nocardioides sp. strain JS614 Noca_0339 (NS), S. coelicolor CRP (SC) (12), C. glutamicum GlxR (CG) (29), M. tuberculosis Rv3676 (MT) (50), Synechocystis sp. strain PCC6803 SYCRP1 (SS) (70), and E. coli CRP (EC) (30) were aligned with the program Clustal W (59). Identical residues in all sequences, conserved substitutions, and semiconserved substitutions are highlighted with red, orange, and yellow, respectively. The e values obtained in the BLAST search are indicated on the right. The cyclic nucleotide monophosphate-binding domain and helix-turn-helix DNA-binding motif identified in the conserved domain database search are indicated.

According to a conserved domain database search, the amino acid sequence of the TTHA1437 protein revealed the presence of two conserved motifs that may be involved in the protein's regulatory activity (Fig. 1). ⁶L to ¹¹⁷H of the TTHA1437 protein comprises a conserved motif that is possibly a cAMP-binding domain, having an e value of 9e-20 with the consensus sequence for cyclic nucleotide monophosphate-binding domains. In addition, a helix-turn-helix DNA binding motif of the CRP/FNR family was identified in the carboxy-terminal region of the TTHA1437 protein (¹⁶²F to ²⁰³R). S. coelicolor CRP, which is the closest homolog of the TTHA1437 protein among the proteins with known functions, has been indicated to bind cAMP, and the residues that are involved in the cAMP binding have been predicted based on a three-dimensional model of the S. coelicolor CRP (12). Based on the amino acid alignment shown in Fig. 1, five of the seven residues, ⁵⁷L, ⁶⁹L, ⁸⁰E, ⁸⁹R, ⁹⁰T, ¹³²R, and ¹³⁵N, which are predicted to bind directly to cAMP are conserved in the TTHA1437 protein (⁵³L, ⁶⁵L, ⁷⁶E, ⁸⁵R, and ¹²⁸R), and one of the two residues, ¹³⁰R and ¹³⁴T, which are predicted to be involved in indirect binding to cAMP, is conserved in the TTHA1437 protein (¹²⁶R).

According to the phylogenetic relationships established by Körner et al. (31), T. thermophilus CRP belongs to the CooA subgroup of the CRP/FNR superfamily (data not shown). However, the residues involved in heme binding in CooA (71) are not present in the TTHA1437 protein, as in cases of S. coelicolor CRP and C. glutamicum GlxR, which also belong to the CooA subgroup, but are suggested to be cAMP receptor proteins (12, 29).

Based on the amino acid sequence properties and the following biochemical characteristics of the TTHA1437 protein, we named this protein T. thermophilus CRP.

Preparation of recombinant T. thermophilus CRP.

The T. thermophilus CRP ORF was cloned under the control of the T7 promoter in E. coli expression vector pET-11a. The initial codon of the ORF found in the chromosome, GTG, was converted to ATG in the expression vector. The CRP gene was overexpressed in E. coli BL21(DE3), and the recombinant protein was purified from a cell lysate. The lysate, which was resistant to treatment at 70°C for 10 min, was precipitated with ammonium sulfate and then fractionated by anion-exchange, hydroxylapatite, and gel filtration column chromatography, resulting in >95% purity on SDS-PAGE (data not shown). We determined the N-terminal amino acid sequence of the purified protein and confirmed that it was recombinant T. thermophilus CRP (data not shown).

To determine whether the T. thermophilus CRP forms a dimer, we performed gel filtration chromatography in the presence of several standards. The CRP was eluted in almost the same fraction as ovalbumin (45.8 kDa) (data not shown). The molecular mass of the CRP estimated on light scattering photometry was 49 kDa (data not shown). These results indicate that T. thermophilus CRP exists as a homodimer in solution.

cAMP-binding ability of T. thermophilus CRP.

T. thermophilus CRP has a conserved motif that is supposed to be a cAMP-binding domain (Fig. 1). To investigate the cAMP-binding ability of T. thermophilus CRP, we performed a protease V8 sensitivity test. In the absence of cAMP, ∼10- and ∼15-kDa fragments derived from T. thermophilus CRP appeared upon treatment with protease V8 (Fig. 2). In the presence of cAMP, an ∼18-kDa band, which was very faint in the absence of cAMP, appeared as one of the major fragments upon V8 digestion (Fig. 2). The protease sensitivity of BSA did not change in the presence or absence of cAMP, and thus cAMP did not inhibit the activity of protease V8 (data not shown). These results suggest that T. thermophilus CRP interacts with cAMP and that this interaction changes the protease sensitivity of the protein. The N-terminal amino acid sequences of the ∼10-, ∼15-, and ∼18-kDa fragments that appeared on V8 digestion are ⁷⁷M-S-L-L-D, ⁷⁷M-S-L-L-D, and ¹M-K-G-S-P, respectively (data not shown). Considering the specificity of protease V8, which digests at the C-terminals of aspartic acid and glutamic acid residues, the ∼10-, ∼15-, and ∼18-kDa fragments possibly correspond to ⁷⁷M to ¹⁶⁸E, ⁷⁷M to ²⁰⁰E and ²⁰¹E, and ¹M to ¹⁶⁸E, respectively. ⁷⁶E of T. thermophilus CRP, which corresponds to one of the predicted cAMP-binding residues (⁸⁰E) of S. coelicolor CRP (12), might directly interact with cAMP, as in the case of E. coli CRP that binds to corresponding ⁷²E (48), resulting in inaccessibility to the protease.

FIG. 2.

Effect of cAMP on the V8 sensitivity of T. thermophilus CRP. Proteolytic digestion of T. thermophilus CRP was performed at 37°C for the indicated periods in the absence (−) (lanes 3 to 6) or presence (+) (lanes 8 to 11) of 1 mM cAMP. Samples were then analyzed by SDS-PAGE. The ∼10-, ∼15-, and ∼18-kDa fragments appearing on V8 digestion are indicated as a, b, and c with arrows. The bands derived from the protease are indicated as V8. NT, nontreated sample; lane 1, molecular mass markers.

Activity of T. thermophilus CRP in vivo.

Using GeneChip technology, we observed the expression profile of T. thermophilus CRP mRNA in vivo during cultivation in a rich medium at 70°C (Fig. 3). Intracellular cAMP was also detected in this strain upon enzyme immunoassay with anti-cAMP antibodies (Fig. 3). The CRP mRNA level and the cAMP concentration varied ∼1.5- and 2-fold, respectively.

FIG. 3.

Three clones of the wild type (closed circles) and those of the ΔCRP strains (open circles) of T. thermophilus were each grown in a rich medium, and the A₆₀₀ values at the indicated times are expressed as means ± SD, respectively. Expression of T. thermophilus CRP mRNA in the wild-type strain at the indicated times (open triangles) was investigated using GeneChip technology, as described in Materials and Methods, and expressed as normalized intensity ± SD. The intracellular cAMP level in the wild-type strain at the indicated times (closed triangles) was monitored by means of a cAMP enzyme immunoassay and expressed as pmol per mg protein ± SD.

To determine the effects of T. thermophilus CRP in vivo, we disrupted the CRP gene of the T. thermophilus HB8 strain and compared the expression level of each mRNA from the mutant with that from that of the wild type. The CRP-disruptant (ΔCRP) strain did not exhibit a growth defect in a rich medium, indicating that this gene is not essential for this strain (Fig. 3). We isolated three wild-type and three ΔCRP strains, respectively, and cultured them for 6 h (A₆₀₀ = 0.8 to 1.0) and 8 h (A₆₀₀ = 3 to 4) in the rich medium, and then the expression of each mRNA was analyzed on a GeneChip as described in Materials and Methods. The expression levels of the CRP mRNA in the ΔCRP strain relative to the wild type after cultivation for 6 h and 8 h were 0.053 (q value = 0.013) and 0.089 (q value = 0.012), respectively, indicating that the CRP gene was disrupted. The data for 33 ORFs could not be obtained, perhaps because inadequate oligonucleotide probes were used for them. From among the 2,205 genes analyzed, we selected those that showed altered expression in the ΔCRP strain with q values of less than 0.05 for both the 6-h and 8-h cultures. In total, eight genes on the chromosomal DNA (designated TTHA) and 22 genes on megaplasmid pTT27 (designated TTHB) were selected (see Table S1 in the supplemental material). All of the selected genes except TTHA0930 and TTHA1574 showed decreased expression in the ΔCRP strain relative to in the wild type (see Table S1 in the supplemental material).

Identification of the target gene of T. thermophilus CRP.

To identify the target gene of T. thermophilus CRP, a runoff transcription assay was performed. We found that DNA derived from the upstream region of the TTHB186 gene exhibiting decreased expression in the ΔCRP strain (Table 1; see also Table S1 in the supplemental material) contained a CRP-dependent promoter. That is, the DNA template containing a 79-bp fragment derived from the upstream region of the TTHB186 gene (genome positions 191068 to 191146 of megaplasmid pTT27) followed by a 125-bp fragment from plasmid pUC19 (Fig. 4A) was hardly transcribed by T. thermophilus RNAP alone; furthermore, the T. thermophilus CRP had no effect on the transcription reaction (Fig. 4B). However, in the presence of the CRP plus cAMP, a transcript was clearly detected (Fig. 4B). These results indicate that the T. thermophilus CRP-cAMP complex can trigger the transcription reaction of T. thermophilus RNAP in vitro.

TABLE 1.

Genes under the control of T. thermophilus CRP-dependent promoters and their expression in the T. thermophilus ΔCRP strain relative to that in the wild type

Gene name	COG no.	Annotation for producta	Expressionb (q value) at time (h)
			6	8
TTHA0176	1670/1247	GCN5-related acetyltransferase	0.772 (0.069)	0.652 (0.107)
TTHA0771	3629	Transcriptional activator of the SARP family	0.291 (0.023)	0.161 (0.106)
TTHB147	1353	Novel DNA polymerase, Csm1 family	0.271 (0.015)	0.149 (0.043)
TTHB148	1421	No prediction, Csm2 family	0.260 (0.015)	0.190 (0.036)
TTHB149	1337	RAMP superfamily, Csm3	0.269 (0.007)	0.294 (0.107)
TTHB150	1567	RAMP superfamily, Csm4	0.302 (0.047)	0.156 (0.020)
TTHB151	1332	RAMP superfamily, Csm5	0.342 (0.013)	0.160 (0.036)
TTHB152		No prediction	0.398 (0.012)	0.260 (0.043)
TTHB156		No prediction	0.030 (0.023)	0.074 (0.073)
TTHB157		No prediction	0.018 (0.021)	0.031 (0.132)
TTHB158		No prediction	0.037 (0.061)	0.052 (0.017)
TTHB159		No prediction	0.596 (0.014)	0.620 (0.180)
TTHB178	2176	Exonuclease III domain	0.154 (0.023)	0.115 (0.012)
TTHB186	2378	Predicted transcriptional regulator	0.063 (0.018)	0.043 (0.012)
TTHB187	1203	CRISPR-associated helicase, Cas3 family	0.101 (0.007)	0.069 (0.020)
TTHB188		Cse1 family	0.252 (0.012)	0.176 (0.020)
TTHB189		Cse2 family	0.191 (0.012)	0.159 (0.036)
TTHB190		Cse4 family	0.241 (0.014)	0.173 (0.036)
TTHB191		Cse5e family	0.219 (0.030)	0.178 (0.036)
TTHB192		Cse3 family	0.159 (0.012)	0.146 (0.037)
TTHB193	1518	Novel nuclease, Cas1 family	0.193 (0.012)	0.143 (0.016)
TTHB194		Cas2 family	0.293 (0.022)	0.176 (0.020)

^aThe annotations for the TTHA0771, TTHB178, and TTHB186 gene products are cited from the descriptions of the corresponding COG families, and those for the TTHA0176, TTHB188, TTHB189, TTHB190, TTHB191, TTHB192, and TTHB194 gene products are from the results of a PSI-BLAST search. The others are cited from Makarova et al. (37, 38), Haft et al. (18), Omelchenko et al. (45), and Brüggemann and Chen (4).

^bNormalized intensity in the ΔCRP strain relative to that in the wild type.

FIG. 4.

(A) Upstream sequence of the TTHB186 gene, which was used as the template for the runoff transcription assays. The numerals represent genome positions in megaplasmid pTT27. The CRP binding region is boxed, and the transcriptional start site is indicated by an asterisk. A possible −10 hexamer sequence of the promoter is indicated by a broken line. (B) Runoff transcription assay, performed with templates a, b, c, and d shown in panel A, in the presence (+) of T. thermophilus CRP (lanes 3, 6, 9, and 12) or the CRP plus cAMP (lanes 4, 7, 10, and 13) or in the absence (−) of both (lanes 2, 5, 8, and 11), as described in Materials and Methods. After the reaction, samples were analyzed by PAGE, followed by autoradiography. Lane 1, [α-³²P]dCTP-labeled MspI fragments of pBR322. (C) Runoff transcription assay, performed with the upstream sequences of TTHB186 (TTHB186_up), TTHB178 (TTHB178_up), TTHB147 (TTHB147_up), TTHA0771 (TTHA0771_up), TTHA0176 (TTHA0176_up), and TTHB159 (TTHB159_up) as templates in the presence of T. thermophilus CRP (lanes 3, 6, 9, 12, 16, and 19) or the CRP plus cAMP (lanes 4, 7, 10, 13, 17, and 20) or in the absence of both (lanes 2, 5, 8, 11, 15, and 18), as described in Materials and Methods. After the reaction, samples were analyzed by PAGE, followed by autoradiography. Lanes 1 and 14, [α-³²P]dCTP-labeled MspI fragments of pBR322.

We found that the region comprising genome positions 191085 of 191107 of megaplasmid pTT27 contains a TCACA block (Fig. 4A and 5B), which is a well-conserved component of the consensus binding sites of CRP family proteins, such as M. tuberculosis CRP_MT (1), P. aeruginosa Vfr (28), X. campestris CLP (22), H. influenzae CRP (7), and E. coli CRP. To determine whether the region is necessary for CRP-cAMP-dependent transcription, we constructed DNA templates of various lengths for in vitro transcription assays (Fig. 4A). The same extent of CRP-cAMP-dependent transcription was observed even when the 5′-terminal 17 bp were deleted from the full-length template containing the 73-bp upstream sequence from the transcription initiation site (Fig. 4B). The efficiency of the transcription was dramatically reduced when an additional 13 bp were deleted from the 5′ terminus; furthermore, transcription was hardly observed when an addition 10 bp were deleted (Fig. 4B). These results suggest that the region necessary for the CRP-cAMP-dependent transcription resides within genome positions 191085 to 191107 of megaplasmid pTT27.

FIG. 5.

(A) Identification of the transcription start site. The RNA transcribed from the genes containing the upstream sequences of TTHB186 (TTHB186_up), TTHB178 (TTHB178_up), TTHB147 (TTHB147_up), TTHA0771 (TTHA0771_up), TTHA0176 (TTHA0176_up), and TTHB159 (TTHB159_up), respectively, in the presence of both T. thermophilus CRP and cAMP, was reverse transcribed with avian myeloblastosis virus reverse transcriptase (lane 5), as described in Materials and Methods. The nucleotide sequence of the template DNA was determined by the dideoxy-mediated chain termination method (lanes 1 to 4). After the reaction, samples were analyzed by PAGE, followed by autoradiography. The 3′ terminus of the cDNA is indicated by an asterisk. (B) Nucleotide sequence alignment of predicted CRP-dependent promoters found in TTHB186_up, TTHB147_up, TTHB178_up, TTHA0771_up, THA0176_up, and TTHB159_up. Possible −10 hexamer sequences of the promoters are indicated by broken lines. The numerals represent the positions from the transcription start site. The conserved bases in the CRP-binding site are boxed, and the consensus sequence (Consensus) is indicated. Nucleotides y, k, m, w, and s represent t or c, g or t, a or c, a or t, and g or c, respectively. Nucleotide n represents g, a, t, or c.

At 22 bp downstream from the TCACA, we found a potential −10 region of the promoter, TAGCAT (Fig. 4A and 5B), which is the same sequence as that of the −10 hexamer of the T. thermophilus-derived 16S rRNA gene (19, 39). These features may represent the CRP-dependent promoter of T. thermophilus. Upstream of the genes and gene clusters that showed altered expression in the ΔCRP strain (q value < 0.05) (see Table S1 in the supplemental material), we searched for sequences that have features similar to those of the possible CRP-dependent promoter of the TTHB186 gene. Among 11 sequences, we found two such sequences upstream of the TTHB178 and TTHB147 genes, the potential −10 hexamers of which have the same sequences as those of the T. thermophilus HB8-derived 23S-5S RNA and 4.5S RNA genes, respectively (19, 39, 56) (Fig. 5B). Furthermore, we searched for the sequence motif upstream of the genes and gene clusters exhibiting altered expression in the ΔCRP strain with at least one of the q values being more than or equal to 0.05 and less than 0.11 (see Table S1 in the supplemental material). Of the 33 sequences we searched, we found three such sequences upstream of the TTHA0771 and TTHA0176 genes and the THB159-158-157-156 gene cluster, respectively (Fig. 5B). Expression of these genes was decreased in the ΔCRP strain relative to expression in the wild type, although one of the q values was higher in the cases of TTHB159 and TTHB157 (Table 1; see also Table S1 in the supplemental material). The potential −10 hexamers of the TTHA0771 and TTHB159 promoters have novel sequences, and that of the TTHA0176 promoter is the same as that of the TTHB186 promoter (Fig. 5B).

To confirm the possibility of CRP-dependent promoters, we performed an in vitro transcription experiment using DNA containing the upstream region of TTHB178, TTHB147, TTHA0771, TTHA0176, or TTHB159 as the template (Fig. 4C). We observed that T. thermophilus CRP enhanced the transcription in a cAMP-dependent manner, indicating that the templates contain a CRP-dependent promoter. Based on nucleotide sequence alignment of the six CRP-dependent promoters, a consensus sequence motif of the predicted T. thermophilus CRP-binding site was proposed (Fig. 5B).

To identify the transcription start sites of the genes, the transcripts synthesized in vitro were reverse transcribed, and then their 3′-terminal nucleotides were identified, respectively (Fig. 5A). The results indicate that the transcription start sites of the TTHB186, TTHB178, TTHB147, TTHA0771, TTHA0176, and TTHB159 genes reside at 34, 30, 30, 30, 33, and 29 bp downstream from the predicted CRP-binding site, respectively (Fig. 5B). In the cases of the TTHB178, TTHB147, and TTHA0771 genes, two major bands were detected as the products of the reverse transcription reaction. In the in vitro transcription experiment involving DNA containing the upstream region of TTHB178, TTHB147, or TTHA0771 as the template, only one major transcript was detected (Fig. 4C); therefore, the lower bands detected on the reverse transcription reaction might represent degradation products of the upper band materials.

To find other possible T. thermophilus CRP-binding sites, we searched for potential binding sites in the whole genome of T. thermophilus HB8 by using a consensus motif of the T. thermophilus CRP-binding site, 5′-GNNCNNNNNTCACA-3′ (N = G, A, T, or C), as a query. As a result, sequences similar to that of the CRP-binding site were found upstream of tRNA-Lys (302915 to 302934 of the chromosomal DNA, 5′-CTTGGCCTATAATCACAAAG-3′), TTHA1221 (1161079 to 1161098 of the chromosomal DNA, 5′-TGGGTCCGGCTTTCACACCC-3′), and TTHB154 (146041 to 146022 of the megaplasmid pTT27, 5′-TCAGGGCCCGGCTCACAACC-3′) genes. However, CRP-cAMP-dependent transcriptional regulation was not observed in an in vitro transcription experiment involving DNA containing 302879 to 302942 or 1161076 to 1161191 of the chromosomal DNA or 146043 to 146074 of megaplasmid pTT27 as the template (data not shown). Upstream of the TTHB142 and TTHA0255 genes, which exhibit decreased and increased expression in the ΔCRP strain (see Table S1 in the supplemental material), respectively, we found similar sequences to those of the CRP-binding sites, but they did not contain the TCACA block, that is, 5′-GGCCCCAAGACCCCACACCT-3′ (128146 to 128165 of megaplasmid pTT27) and 5′-AGCGTTGCGTTTGCACACCC-3′ (242544 to 252557 of the chromosomal DNA). However, the effect of the CRP-cAMP on in vitro transcription was not observed when DNA containing 128143 to 128203 of megaplasmid pTT27 or 242544 to 252598 of the chromosomal DNA was used as the template (data not shown).

Table 1 summarizes the genes under the CRP-dependent promoter found in this study and their altered expression levels in the ΔCRP strain relative to those in the wild type.

DISCUSSION

Extremely thermophilic bacterium T. thermophilus HB8, of the phylum Deinococcus-Thermus, possesses a member of the CRP family, T. thermophilus CRP, which is highly homologous to the CRP homologs from strains that belong to the phyla Firmicutes, Actinobacteria, and Cyanobacteria. T. thermophilus CRP forms a homodimer and interacts with cAMP. The cAMP-binding domain of T. thermophilus CRP is highly conserved not only in the homologs with higher e values but also in ones with lower e values, but it is phylogenetically distant. Therefore, the cAMP interaction with CRP might be similar among the CRP family proteins. The structural changes in CRP caused by cAMP binding have not been elucidated at the atomic level because the crystal structure of CRP without cAMP has not been determined yet. Many T. thermophilus proteins are quite stable, and thus, T. thermophilus CRP is a good candidate for such X-ray crystallographic studies.

Of the genes exhibiting altered expression in the T. thermophilus ΔCRP strain, we identified six CRP-dependent promoters upstream of the TTHB186, TTHB147, TTHB178, TTHA0771, TTHA0176, and TTHB159 genes, respectively. Expression of genes downstream of the TTHB186, TTHB147, and TTHB159 ones were also decreased in the ΔCRP strain (Table 1); therefore, they may form operons, respectively, as indicated by the genome analysis (NCBI accession numbers NC_006461, NC_006462, and NC_006463).

Based on nucleotide sequence alignment of the six CRP-dependent promoters from T. thermophilus HB8, a consensus sequence of the predicted CRP-binding site, 5′-(C/T)NNG(G/T)(G/T)C(A/C)N(A/T)NNTCACAN(G/C)(G/C)-3′, was proposed. This sequence is unique compared with the known binding sequences of closer homologs of T. thermophilus CRP, such as C. glutamicum GlxR (36) and M. tuberculosis CRP_Mt (1), as well as those of phylogenetically distant homologs such as E. coli CRP, P. aeruginosa Vfr (28), X. campestris CLP (22), and H. influenzae CRP (7). The crystal structure of the E. coli CRP-DNA complex revealed that one subunit of the CRP dimer binds to the left arm ⁴TGTGA block, while the other binds to the right arm ¹⁵TCACA block of the 22-bp consensus CRP-binding site, and the ¹⁸⁰R, ¹⁸¹E, and ¹⁸⁵R residues of E. coli CRP directly interact with the G:C pairs at positions 5 and 7 and the A:T pair at position 8 of the consensus CRP-binding half site, respectively (47, 52). The manner of the DNA recognition by T. thermophilus CRP may differ from that in the case of E. coli CRP because the left arm of the consensus T. thermophilus CRP-binding site differs from that of E. coli CRP, although the right arm block, ¹³TCACA, might correspond to the ¹⁵TCACA block of the E. coli consensus CRP-binding site. It should be noted that S. coelicolor CRP, one of the closer homologs of T. thermophilus CRP, did not recognize a DNA sequence similar to that of the E. coli CRP-binding site (12). The structure of the C-terminal DNA-binding domain of T. thermophilus CRP might differ from that of E. coli CRP, as predicted in the case of S. coelicolor CRP (12), even though ¹⁸⁰R, ¹⁸¹E, and ¹⁸⁵R of E. coli CRP, which directly interact with DNA, are conserved in T. thermophilus CRP (¹⁷⁷R, ¹⁷⁸E, and ¹⁸²R).

The mechanism underlying the transcriptional activation by CRP has been precisely studied in E. coli. In E. coli, simple CRP-dependent promoters, which require one CRP dimer for transcription activation, are grouped into two classes, I and II, based on the position of the CRP-binding site and the transcription activation mechanism (2, 6, 34). In the class I CRP-dependent promoters, the CRP-binding site is located upstream of the core promoter, centered at position −61.5 from the transcription start site. When CRP binds to this site, its activation region 1 (AR1) comes into contact with RNAP's αCTD to recruit RNAP to the promoter (2, 34). In the class II CRP-dependent promoters, the CRP-binding site is centered at position −41.5, which overlaps the core promoter −35 element, and contact occurs between CRP's AR1, AR2, and AR3, and RNAP's αCTD, αNTD, and σ⁷⁰, respectively. In the cases of the T. thermophilus CRP-dependent promoters found in this study, the CRP-binding sites reside at position −38 to −43 from the transcription start site for the promoters, and the −35 element of these promoters may overlap the CRP-binding site. Thus, these promoters are more similar to the class II than the class I CRP-dependent promoters in E. coli.

To determine the cellular role of the T. thermophilus CRP, we had a look at the amino acid sequence features of the products of the CRP-regulated genes because the gene products have been biochemically or biophysically uncharacterized. Upstream (genome positions 189507 to 190947 of megaplasmid pTT27) and downstream (genome positions 200811 to 202078 of megaplasmid pTT27) of a probable operon, TTHB186-187-188-189-190-191-192-193-194, imperfect short palindromic sequences called clustered regularly interspaced short palindromic repeats (CRISPRs), which are found in various prokaryotic cells (25-27, 41, 42, 53), were found. Therefore, these genes have been called CRISPR-associated (cas) genes (17, 18, 26), and their products have been hypothesized to be thermophile-specific DNA repair systems or prokaryotic host defense systems against invading foreign replicons because some of the gene products have some features similar to those involved in nucleotide metabolism (4, 14, 17, 18, 38, 45). A probable operon, TTHB147-148-149-150-151-152, is preceded and followed by another type of CRISPR; genome positions 135156 to 136099, 144129 to 144842, and 146042 to 146905 of megaplasmid pTT27. The products of this type of cas gene except TTHB152 are classified as the Mtube subtype (18) and are also predicted to be thermophile-specific DNA repair systems or host defense systems (4, 18, 38, 45).

The TTHB178 protein belongs to COG2176 and contains the exonuclease III domain found in exonuclease proteins, such as the ɛ subunit of DNA polymerase III that shows 3′ to 5′ exonuclease activity, and RNase T that is responsible for removing the terminal AMP residues from uncharged tRNA (4).

The TTHA0771 protein belongs to COG3629 and is classified as a DNA-binding transcriptional activator of the Streptomyces antibiotic regulatory protein (SARP) family, the N-terminal domain of which exhibits homology with the C-terminal DNA-binding domain of OmpR family proteins (63).

The TTHA0176 protein belongs to the GCN5-related acetyltransferase family, which is an enormous superfamily of enzymes that are universally distributed in nature and that use acyl coenzyme A to acylate their cognate substrates (62).

According to a BLAST search, only poorly conserved proteins were found using the TTHB156, TTHB157, TTHB158, or TTHB159 protein as a query, suggesting that these proteins are T. thermophilus-specific proteins of unknown function.

In T. thermophilus HB8, the aforementioned genes are basically expressed under the experimental conditions used. The genes that showed altered expression in the ΔCRP strain might include those that are not directly regulated by CRP but are affected by the gene products which are under direct control of CRP. The expression levels of the CRP-regulated genes are supposed to change when some environmental changes that lead to changes in the intracellular cAMP level occur. T. thermophilus HB8 has a gene encoding an adenylate cyclase-like protein, TTHA1259 (NCBI accession number YP_144525), implying that signal transduction involving cAMP occurs in this strain. Identification of such signals may shed light on the functions of these gene products and the cellular role of this regulator in the physiology of T. thermophilus.