1 genome wide identification of targets for the archa...

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GENOME WIDE IDENTIFICATION OF TARGETS FOR THE ARCHAEAL HEAT 1

SHOCK REGULATOR PHR BY CELL FREE TRANSCRIPTION OF GENOMIC DNA 2

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Annette M. Keese1, Gerrit J. Schut

2, Mohamed Ouhammouch

3, Michael WW. Adams

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Michael Thomm1* 5

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1 Department of Microbiology, University of Regensburg, Universitaetsstr. 31, D-93053 7

Regensburg, Germany 8

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2 Department of Biochemistry, A 214 Life Sciences Building, Athens, Georgia 30602 – 7229, 10

U.S.A. 11

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3 Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, 13

La Jolla, CA 92093-0634, U.S.A. 14

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Running title: Novel archaeal heat shock genes 17

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* Corresponding author Michael Thomm 19

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Correspondent footnote: 21

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Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00924-09 JB Accepts, published online ahead of print on 18 December 2009

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The hyperthermophilic archaeon, Pyrococcus furiosus, grows optimally near 100°C 35

and undergoes a heat shock response at 105°C mediated at least in part by the heat 36

shock regulator Phr. Genes encoding a small heat shock protein (HSP20) and a member 37

of the AAA+ ATPase are the only known targets of the regulator, but a genetic mutant of 38

Phr has yet to be characterized. We describe here an alternative approach for the 39

identification of the regulon of Phr based on cell-free transcription of fragmented 40

chromosomal DNA in the presence or absence of the regulator and hybridization of in 41

vitro RNA to P. furiosus whole genome microarrays. Our results confirmed the phr, the 42

hsp20 and the aaa+ atpase genes as targets of Phr and also identified six additional open 43

reading frames, PF0624, PF1042, PF1291, PF1292, PF1488 and PF1616, as Phr 44

responsive genes which include that encoding di-myo-inositol phosphate synthase. 45

Transcription of the identified novel genes was inhibited by Phr in standard 46

transcription assays and the novel consensus sequence 5´-47

TTTAnnnACnnnnnGTnAnnAAAA-3´ was inferred from our data as the Phr 48

recognition motif. Mutational evidence for the significance of this sequence as Phr 49

recognition was provided in DNA binding experiments. 50

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INTRODUCTION 69

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In archaea, proteins of the AAA+family of ATPases (AAA

+-proteins), small heat shock 71

proteins (sHSP) and the β1 subunit of the proteasome are the only known proteins whose 72

corresponding genes are induced upon heat shock (7, 10). HSP20 is the best studied sHSP. It 73

is conserved among hyperthermophilic and thermophilic Euryarchaeota (e.g. Pyrococcus, 74

Methanocaldococcus, Methanobacterium), hyperthermophilic (e.g. Aquifex) and mesophilic 75

(e.g., Clostridium) bacteria and is also found in eukaryotes (e.g. Oryza sativa) (6, 5, 7). Hsp20 76

has been most studied in the hyperthermophile Pyrococcus furiosus. Its production is 77

stimulated, both at the mRNA and protein levels, by raising the growth temperature from 95 78

to 105 °C, (6) and its function as a heat shock protein has been established. Hsp20 prevents 79

proteins from mesophilic organisms from aggregating up to a temperature of 105 °C in vitro 80

and increased survival of E. coli cells at 50 °C for 2h compared with control cultures, when 81

expressed from an inducible promoter (6). 82

While the role of the AAA+ proteins in the heat shock response is still unclear, sHSP seem 83

to function mainly as holdases preventing denaturation and aggregation of proteins at elevated 84

temperatures (6). In addition, homologues of the Hsp60 family are present in the genomes of 85

all archaea and upregulation of their upregulation as part of multiprotein complexes known as 86

thermosomes during stress response has been demonstrated (21, 17). The archaeal 87

thermosomes form large homooligomeric rings consisting of 7-9 subunits that assist in protein 88

folding as well as in other functions (14, 4, 20). Genomic mining revealed also the presence of 89

prefoldin homologues in all archaeal species studied so far but the gene encoding the 90

prefoldin β-subunit was down regulated in Pyrococcus upon heat shock (17). A role of 91

archaeal prefoldins in heat shock response remains to be established. On the other hand, 92

archaea lack homologs of the Hsp 70, Hsp90 and Hsp100 families of chaperones (7). Thus, 93

hyperthermophiles were proposed to possess simplified heat shock systems which might 94

represent the minimal folding system present in the early eukaryotes (7). It is also possible 95

that hitherto unknown heat shock proteins as well as other response mechanisms contribute to 96

protein refolding and stabilization exist in archaea. 97

In the hyperthermophile Pyrococcus furiosus, a transcriptional regulator of heat shock 98

proteins, termed Phr, has been identified and characterized in some detail (22; 8). Phr binds 99

specifically to promoters of genes encoding the small heat shock protein Hsp20 and the AAA+

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protein. Phr also binds to a region overlapping the transcription start site of its own gene, both 101

in vitro and in vivo. At the physiological growth temperature of 95 °C, Phr was shown to 102


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associate with these promoters in P. furiosus cells but was released when the temperature was 103

increased to 107 °C (8). Thus, expression of heat shock genes seems to correlate with 104

dissociation of Phr from heat shock promoters. The cellular mechanism directing release of 105

Phr at elevated temperatures is unknown. DNA-binding experiments revealed that Phr binding 106

prevents RNA polymerase recruitment to heat shock promoters and mutational analyses 107

suggested that a TTTA motif at -10 is essential for binding of Phr to heat shock promoters in 108

vitro (22). 109

Cell-free transcription of cleaved chromosomal DNA has been used previously as a tool to 110

identify the genes targeted by a specific transcriptional regulator in bacteria (1, 9, 23). Briefly, 111

restricted chromosomal DNA is used as template in cell-free transcription assays in the 112

presence or absence of a transcriptional regulator. The RNA produced in vitro is then labeled 113

and hybridized to a whole genome microarray to determine changes in the amount of any 114

RNA species due to the presence of the regulator. In this manner, the genes that are up-or 115

down-regulated by the specific regulator can be identified. We have used this approach 116

herein with P. furiosus Phr and have identified six previously unknown archaeal heat shock 117

genes and a novel DNA motif characteristic of P. furiosus heat shock genes. 118

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MATERIALS AND METHODS 122

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Whole genome in vitro transcription. SmaI-digested chromosomal DNA from P. furiosus 124

was used as template for cell-free transcription experiments (independent digest in each 125

experiment shown in Table 1). SmaI was selected among several restriction enzymes because 126

it generates blunt ends which are unlikely to facilitate initiation at ends of DNA fragments. 127

Transcription reaction mixtures (100 µl) contained 40 mM NaHEPES, pH 7.3; 300 mM NaCl; 128

6 mM MgCl; 0.1 mM EDTA; 0.2 mM DTT and 0.01mg/ml BSA. The transcription reactions 129

contained 120 nM TFB (recombinant PF1377 – 3); 120 nM TBP (recombinant PF1295 – 3); 130

262 nM RNA polymerase (RNAP; 3); 1 mM ATP; 1 mM GTP; 0.4 mM CTP and 0.1 mM 131

UTP. (ATP and GTP are initiator nucleotides at archaeal promoters and were added in 10 132

fold excess over UTP/CTP to facilitate initiation). For the transcription reaction, chromosomal 133

DNA (65 pM), with and without recombinant Phr (2.7 µM, PF1790, 21), were incubated for 134

10 min at 70°C and the transcription factors and RNAP were added. After a further incubation 135

for 10 min at 70°C, the transcription was started by adding the nucleotides. After 30 min the 136

reaction was stopped by phenol/chloroform/isoamylalcohol extraction. RNA was purified by 137

DNase I digestion, phenol/chloroform/isoamylalcohol extraction and filtration using 138

Microcon Ultracel YM-30 according to the manufacturer`s instructions (Millipore). 139

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RNA labeling and microarray analyses. 1.5 µg RNA from in vitro transcription was 141

labelled with Alexa dye 647 or 594 using ULYSIS Nucleic Acid Labeling Kits (Invitrogen, 142

Carlsbad, CA) according to the manufacturer’s instructions. The labeled RNA was purified 143

using Micro Bio-Spin 30 Columns (Bio-Rad) and dried under vacuum. Differentially labelled 144

RNAs from basal genomic in vitro transcription and genomic in vitro transcription in presence 145

of Phr were pooled and hybridized to the P. furiosus DNA microarray. The fluorescence 146

intensities for the Alexa dyes were measured by using a Scan Array 5000 spectrometer 147

(PerkinElmer Waltham, MA) with the appropriate Sand filter settings and analysed by using 148

Quantarray (PerkinElmer). To determine which genes are affected by the presence of Phr we 149

used scatterplot comparisons of the relative fluorescent intensities. Those that deviated from 150

the 1:1 ratio of fluorescent intensities (diagonal in Fig: 1) were selected for further studies 151

using in vitro transcription analysis. 152

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Standard in vitro transcription. The in vitro transcription was performed as described 154

previously (3). Templates were PCR-amplified from chromosomal DNA. The transcription 155


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reactions were carried out in a final volume of 50 µl, and contained 40 mM NaHEPES, pH 156

7.3; 6 mM MgCl2; 0.1mM EDTA; 300 mM NaCl; 0.44 mM each of ATP, GTP CTP, 0.002 157

mM UTP and 2 µCi[α-32

P]-UTP; 23 nM TFB (recombinant); 104 nM TBP (recombinant), 19 158

nM RNAP and varying concentrations (0-200 nM) of recombinant Phr. The reactions were 159

incubated for 30 min at 70°C. Run-off transcriptions were analyzed on a denaturing 8% 160

polyacrylamide-gel quantified and visualised using a PhosphoImager (FLA-5000, Fuji, Japan). 161

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Primer extension analysing transcription from chromosomal DNA. The end-labelled 163

primers used for the reactions were as follows: 5`-GCAGGAATAGTATTCTTCTCC-3` 164

complementary to nucleotides +50 to +70 of phr, 5`-CCTAGCTCTCTCATCGTTTCC-3` 165

complementary to nucleotides +87 to +107 of aaa+atpase, 166

5`-GCTGAAGAATTCATCGAACATTGC-3`complementary to +76 to +99 nucleotides of 167

hsp20 and 5`-GCTTCTTCACTTATCTCC-3` complementary to nucleotides +62 to +80 of 168

gdh. The primers were annealed with RNA from whole genome in vitro transcription and 169

extended with reverse transcriptase. The cDNA was analyzed on an 8% polyacrylamide urea 170

gel in 1 x TBE. Primer extension products were visualized by phosphoimaging (FLA-5000, 171

Fuji, Japan). 172

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EMSA. The reactions were assembled in a final volume of 10 µl. Each reaction contained 40 174

mM HEPES-NaOH, pH 7.3, 325 mM NaCl, 2.5 MgCl2, 0.1 EDTA, 5% polyethylene glycol 175

(PEG) 8000, 3µg of bovine serum albumin and 1 µg of poly(d(IC)) as non-specific competitor 176

DNA. Binding reactions contained 2.1 µM DNA and 10 µM Phr and were incubated at 70°C 177

for 10 min. DNA protein complexes were analysed on nondenaturing 15 % acrylamide gels. 178

The DNA was stained with ethidium bromide and visualised using a PhosphoImager (FLA-179

500, Fuji, Japan). 180

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RESULTS 182

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Identification of Phr targets by the use of microarrays. SmaI digested chromosomal 184

DNA (average size of fragments ~17 kb) was transcribed in vitro assays carefully optimized 185

as indicated in Materials and Methods. Reactions were conducted in the presence or absence 186

of recombinant Phr (PF1790). In particular, the Phr to DNA ratio was optimized using a 187

primer for the hsp20 heat shock gene. The result of the primer extension analysis showed that 188

a molar concentration of Phr between 2.1 to 2.7 µM was sufficient for almost complete 189

repression of this heat shock promoter (supplementary Fig. 1). For the identification of 190

unknown Phr binding sites, 2.7 µM Phr was added to all transcription reactions with genomic 191

DNA described here. The resulting RNA was fluorescence-labelled and hybridized to P. 192

furiosus whole genome microarrays (16; Fig. 1). In five independent experiments, nine P. 193

furiosus ORFs were found to be significantly down-regulated by Phr (Tab. 1). The gene 194

encoding Hsp20 (PF1883) was identified as a Phr target in all five experiments, while the 195

aaa+atpase gene (PF1882) and the phr gene (PF1790) were down regulated by Phr in three 196

and two experiments, respectively (Table 1). Transcription of the genes phr, hsp20, 197

aaa+atpase and gdh (encoding glutamate dehydrogenase, used as a control) was probed by 198

primer extension using gene specific primers (Figure 2). These findings demonstrated that 199

transcription regulation by Phr is readily detected in our in vitro system utilizing 200

chromosomal DNA as template, and suggests that this approach could be used to identify 201

additional targets of Phr regulation. Indeed, beside the known heat shock genes listed above, 202

transcription of six additional open reading frames was found to be affected by Phr. PF0624 203

was shown to be down-regulated by Phr in four experiments, PF1488 and PF1616 in five, 204

PF1292, PF1042 in two and PF1291 in one experiment (Tab. 1). These data suggest that the 205

Phr regulon is comprised of at least 9 potential targets. Three of these (PF1790, PF1882, 206

PF1883) were known from previous studies (22) but six new Phr sensitive genes (PF0624, 207

PF1042, PF1291, PF1292, PF1488 and PF1616) have been discovered by this study. 208

Five of these genes were not detected as heat shock responsive genes in a previous 209

microarray based study (17). In these experiments only ~ 10 percent of the Pyrococcus 210

genome was analyzed and the particular genes identified here were not included. In contrast, 211

PF1616 encoding the myo-inositol-phosphate synthase was identified as heat shock gene by 212

Northern blotting (17). 213

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Verification of six novel targets of Phr by in vitro transcription. To determine whether 215

the six genes identified as putative targets for Phr are repressed in vitro by Phr, standard cell-216

free transcription analyses were performed using the promoter regions of PF0624, PF1042, 217

PF1291, PF1292, PF1488 and PF1616 as well as the known targets PF1882, PF1883 and 218

PF1790. These promoter regions were amplified by PCR and used as templates in in vitro 219

transcription assays carried out in the presence or absence of Phr. Transcription initiation 220

from all these promoters was inhibited by the addition to the transcription reaction of 221

increasing concentrations of Phr (Fig. 3A and B). In reactions containing a Phr to template 222

ratio of 20 (last lane in each panel in Fig. 3A) the residual transcription signals ranged from 5-223

56% of the control lacking Phr. For the previously characterized P. furiosus heat shock genes 224

aaa+atpase, hsp20 and the gene encoding Phr itself (phr), the residual activity at a Phr to 225

DNA ratio of 20 was 17-26% (Fig. 3B). Thus, two of the novel promoters (PF0624 and 226

PF1042) were inhibited more effectively by Phr, two (PF1291 and PF1488) to a similar extent 227

and at two promoter sites (PF1292 and PF1616) inhibition by Phr was weaker than at known 228

heat shock genes (compare last lanes in Fig. 3A and B). The lower sensitivity of PF1616 can 229

be also caused by the presence of two Phr binding sites at this promoter, which were both 230

bound by Phr as shown by gel shift analyses (see supplementary Fig. 3). Even at a Phr to 231

DNA ratio of 10, the transcription of all novel Phr targets was significantly impaired (see 232

third lane in Fig. 3A and B). The residual activities were ~80% for PF1616, PF1291 and 233

PF1292, ~50% for PF1488 and PF1042, and ~20% for PF0624. By contrast, expression of the 234

gdh promoter used as a control was not affected significantly by Phr even at high Phr to 235

template ratios. These data therefore unequivocally identify the six novel genes as being 236

regulated at the transcriptional level by Phr in vitro. 237

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Identification of a consensus sequence for Phr regulated genes. The discovery of novel 239

target genes for Phr in the genome of P. furiosus enabled a further characterization of the 240

consensus promoter sequence recognized by this regulatory protein. We aligned the upstream 241

regions of the genes affected in vitro by Phr. (Fig. 4A). For eight of them (PF0624, PF1042, 242

PF1291, PF1488, PF1616, PF1790, PF1882 and PF1883) there was a perfect conservation of 243

the bases (denoted by uppercase in the following sequence): 244

TTTAnnnAcnnnnnGTnAnnAAAa (Fig. 4A). In addition, two bases (c at position 9 and at the 245

last position, shown in lower case) of the consensus were conserved in seven of the eight 246

promoter regions. When all promoter regions were aligned using MEME (18), a perfect 247

conservation of three T-residues at the 5´end of the consensus, of the GT motif at positions 15, 248


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16 of an A at position 18 and a high conservation of two A residues at positions 22 and 23 of 249

the consensus was found (Fig. 4C). The alignment of all Phr regulated genes identified the 250

sequence TTT(A/T)ntn(A/T)(A/C)nnnnnGTnAnn(A/T)AAa as the consensus binding 251

sequence for Phr. This consensus sequence was then used to query a database containing all 252

P. furiosus promoter regions using the MAST Program (19). This analysis identified PF0321 253

as additional potential Phr target. All Phr binding sites with an E-value < 0.074 254

(supplementary Table 1) were identified by microarray analyses with exception of the binding 255

site upstream of PF0321. This gene contains two TATA boxes in tandem and two in frame 256

ATG initiator codons (supplementary Fig. 2B). In vitro transcription analyses from this 257

promoter revealed that two transcripts were formed, the longer transcript most likely 258

expressed from the more upstream TATA box was inhibited at high Phr concentrations. But at 259

high Phr concentrations the shorter transcript expressed from the more downstream TATA 260

box was still formed (Fig. 2A) and the result that only one of the two PF0321 promoters was 261

turned off at high Phr concentrations explains that this gene was not identified as Phr binding 262

site in the microarray studies. Because the existence of two transcription start sites for this 263

gene was not yet analyzed and confirmed in vivo, PF0321 was not included into the statistical 264

analysis conducted to infer the Phr consensus sequence. PF0321 encodes a putative inosine 265

monophosphate dehydrogenase. 266

No additional Phr targets could be identified in the genome. The complete consensus 267

sequence is only present in the promoter of the nine (ten) identified genes. 268

To provide biochemical evidence for the significance of the consensus sequence a series of 269

single point mutations in the Phr binding sequence was generated and the effect of mutations 270

on Phr binding was assayed in electrophoretic mobility shift assays. Strong binding of Phr 271

was observed to a synthetic ds oligonucleotide containing the consensus sequence which was 272

found upstream of the gene coding PF0624 (Fig. 5, lanes 1 and 2). When 13 positions of the 273

consensus were changed as shown in the sequence A below the Figure, no binding of Phr 274

occurred (Fig. 5, lanes 3 and 4). When the ttta partial palindromic sequence at the left side of 275

the consensus was replaced by gggc or when the aaaa sequence at the right end of the 276

consensus sequence was changed to gggg, binding of Phr was greatly impaired (Fig. 5, lanes 277

5,6 and 9,10) indicating that these flanking palindromic sequences are important for Phr 278

binding. In addition, mutation of 5 conserved residues in the central part of the Phr consensus 279

sequence (see sequence shown in C below the Figure) resulted in significantly reduced 280

binding of Phr. Taken together these results support the conclusion that the palindromic 281

flanking regions of the consensus sequence are most important for Phr binding, but that the 282


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conserved sequences in the central part of the binding site contribute in addition to Phr 283

binding. 284

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Physiological implication of the Phr regulon. Of the six new genes that we discovered 286

were regulated by Phr (Tab. 1), one of them (PF1616) was identified previously as a heat 287

shock-responsive gene (17). PF1616 encodes the enzyme myo-inositol-phosphate synthase 288

and is involved in the synthesis of the compatible solute di-myo-inositol-1,1´-phosphate (DIP). 289

DIP has been shown to accumulate to high molar concentrations in the cytoplasm of P. 290

furiosus at suboptimal growth temperatures. It has been shown to stabilize proteins at 291

extreme temperatures in vitro and has been proposed to afford similar protection in vivo (15, 292

11, 2). Three of the other five new Phr-regulated genes listed in Table 1 are annotated as 293

encoding conserved hypothetical proteins (PF0624, PF1292 and PF1488). Of these, PF1292 294

only has homologs in bacteria (mainly Firmicutes), suggesting that it arose in P. furiosus by 295

lateral gene transfer, while PF0624 and PF1488 have homologs in both the archaeal and 296

bacterial domains. Of the other two genes regulated by Phr, PF1291 has homologs only in the 297

genomes of the Euryarchaeota. It encodes a phosphoesterase of unknown specificity, perhaps 298

involved in the synthesis of the organic phosphates that are also synthesized during heat shock 299

of P. furiosus (11). Interestingly, the adjacent genes PF1291 and PF1292 are both regulated 300

by Phr but are transcribed in opposite directions. The other gene, PF1042, is annotated as a 301

member of the ferritin/ribonucleotide-like family but this offers no clue to its function since 302

the prototypical members (ferritin, ribonucleotide reductase and ruberythin) have all been 303

characterized from P. furiosus. 304

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DISCUSSION 306

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The combination of run-off transcription of chromosomal DNA followed by hybridization 308

to macro- or microarrays (ROMA) has been successfully established as a method to identify 309

promoters regulated by σw in Bacillus subtilis (1) and to identify promoters targeted by the 310

cAMP-receptor protein in Escherichia coli (23). In archaea, experiments using chromosomal 311

DNA of Methanocaldococcus jannaschii as template revealed that activation of transcription 312

by the transcriptional regulator Ptr2 can be observed not only when short DNA fragments 313

were used, but also when all potential competitive binding sites of the regulator were present 314

on the template consisting of genomic DNA (12,13). On the basis of these findings, the 315

potential of this approach to identify novel binding sites of the archaeal heat shock regulator 316

Phr of P. furiosus was analyzed in this study. Several lines of evidence indicate that the 317

ROMA approach described here can be used to identify a significant fraction of the Phr 318

regulon and probably also targets of other archaeal regulators. First, the heat shock genes hsp 319

20 and aaa+atpase established as Phr-binding sites in vitro and in vivo (22, 8) were shown to 320

be inhibited by Phr in both our ROMA experiment (Fig. 1 and Table 1) and when 321

chromosomal DNA was used as template in optimized cell-free transcription reactions (Fig. 2). 322

Second, the six novel Phr targets identified by the ROMA approach were all inhibited by Phr 323

in subsequent standard cell-free transcription reactions from promoters located on short DNA 324

fragments (Fig. 3). Third, all identified Phr promoters shared a common highly conserved 325

sequence (Fig. 4) which is proposed here as consensus sequence of promoters regulated by 326

Phr. 327

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Our study confirms the results of a previous study obtained using a DNA microarray in 329

conjunction with Northern blotting (17) that the genes encoding the AAA+ VAT type of 330

ATPase (PF1882) and di-myo-inositol 1-phosphate synthase (PF1616) were up-regulated in P. 331

furiosus upon heat shock. However, that study also identified the gene encoding the major 332

Hsp60-like chaperonin as a heat-inducible protein but the results presented here show that this 333

gene (PF1974) is not regulated by Phr. Accordingly, the hsp60 promoter region does not 334

contain the consensus Phr-binding site motif (Fig. 4C and supplementary Table 1) and the 335

gene is not regulated by Phr in vitro (Vierke, G. and Thomm, M, unpublished data). The same 336

is presumably true for the more than 20 additional genes that were up-regulated by heat shock 337

(17). These findings indicate that heat shock regulation in P.furiosus is complex and can be 338

mediated by a different mechanism on many promoters without involvement of Phr. Our 339


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finding that 5 genes hitherto not assigned to encode heat shock proteins in P. furiosus, 340

PF0624, PF1042, PF1291, PF1292 and PF1488, shows that a single approach is insufficient to 341

identify all genes involved in stress response or targets of a given regulator. As previously 342

pointed out (9), a combination of complementary approaches is needed to obtain maximal 343

coverage when defining targets of a regulator. 344

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None of the novel Phr-regulated genes identified in this study had homologs in eukaryotic 346

genomes (Tab. 1). Therefore, it is unlikely that they contribute to the pool of primordial heat 347

shock genes shared between the ancestor of archaea and eukaryotes. As indicated in Table 1, a 348

putative function can be clearly assigned to PF1291 as a putative phosphodiesterase. PF0321 349

is related to a inosine monophosphate dehydrogenase. It remains to be determined exactly 350

which role the additional identified key proteins play in stress reponse. Furthermore the 351

primary response of this hyperthermophilic organism to the fundamental physiological 352

response of being exposed to a suboptimal growth temperature still has to be elucidated.. 353

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ACKNOWLEDGMENTS 356

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This research was supported by grants from the US Department of Energy (FG05-358

95ER20175 and FG02-08ER64690 to MWA) and by the priority program “Genome 359

Organization and Regulation of Genome Expression in Archaea” funded by the Deutsche 360

Forschungsgemeinschaft (to MT). 361

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REFERENCES 364

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1. Cao, M., P. A. Kobel, M. M. Morshedi, M. F. W. Wu, C. Paddon, and J. D. Helmann. 366

2002. Defining the Bacillus subtilis σw regulon: A comparative analysis of promoter 367

consensus search, run-off transcription/macroarray analysis (ROMA), and transcriptional 368

profiling approaches. J. Mol. Biol. 316:443-457. 369

2. Hensel, R., and H. König. 1988. Thermoadaptation of methanogenic bacteria by 370

intracellular ion concentration. FEMS Microbiol. Lett. 49:75-79. 371

3. Hethke, C., A. C. M. Geerling, W. Hausner, W. de Vos, and M. Thomm. 1996. A cell-372

free transcription system for the hyperthermophilic Archaeon Pyrococcus furiosus. 373

Nucleic Acids Res. 25:2369-2376. 374

4. Klumpp, M., and W. Baumeister. 1998. The thermosome: archaetype of group II 375

chaperonins. FEBS Lett. 430:73-77. 376

5. Laksanalamai, P., A. Jiemjit, Z. Bu, L. Maeder, and F. T. Robb. 2003. Multi-subunit 377

assembly of the Pyrococcus furiosus small heat shock protein is essential for cellular 378

protection at high temperature. Extremophiles. 7:79-83. 379

6. Laksanalamai, P., D. L. Maeder, and F. T. Robb. 2001. Regulation and mechanism of 380

action of the small heat shock protein from the hyperthermophilic archaeon Pyrococcus 381

furiosus. J. Bacteriol. 183:5198-5202. 382

7. Laksanalamai, P., T. A. Whitehead, and F. T. Robb. 2004. Minimal protein-folding 383

systems in hyperthermophilic Archaea. Nature Rev. Microbiol. 2:315-324. 384

8. Liu, W., G. Vierke, A.-K. Wenke, M. Thomm, and R. Ladenstein. 2007. Crystal 385

structure of the archaeal heat shock regulator from Pyrococcus furiosus: a molecular 386

chimera representing eukaryal and bacterial features. J. Mol. Biol. 369:474-488. 387

9. MacLellan, S. R., W. Eiamphungporn, and J. D. Helmann. 2009. ROMA: An in vitro 388

approach to defining target genes for transcription regulators. Methods 47:73-77. 389

10. Madding, L. S., J. K. Michel, K. R. Shockley, S. B. Conners, K. L. Epting, M. R. 390

Johnson, and R. M. Kelly. 2007. Role of the β1 subunit in the function and stability of 391

the 20S proteasome in the hyperthermophilic archaeon Pyrococcus furiosus. J. Bacteriol. 392

189:583-590. 393

11. Martins, L. O., R. Huber, H. Huber, K. O. Stetter, M. S. Da Costa, and H. Santos. 394

1997. Organic solutes in hyperthermophilic Archaea. Appl. Environ. Microbiol. 63:896-395

902. 396


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http://jb.asm.org/

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12. Ouhammouch, M., R. E. Dewhurst, W. Hausner, M. Thomm, and E. P. Geiduschek. 397

2003. Activation of archaeal transcription by recruitment of the TATA-binding protein. 398

Proc. Natl. Acad. Sci. USA. 100:5097-5102. 399

13. Ouhammouch, M., G. E. Langham, W. Hausner, A. J. Simpson, N. M. El-Sayed, and 400

E. P. Geiduschek. (2005) Promoter architecture and response to a positive regulator of 401

archaeal transcription. Mol. Microbiol. 56:625-37. 402

14. Quaite-Randall, E., J. D. Trent, R. Josephs, and A. Joachimiak. 1995. Conformational 403

cycle of the archaeosome, a TCP1-like chaperonin from Sulfolobus shibatae. J. Biol. 404

Chem. 270:28818-28823. 405

15. Scholz, S., J. Sonnenbichler, W. Schäfer, and R. Hensel. 1992. Di-myoinositol-1,1’-406

phosphate: a new inositol phosphate isolated from Pyrococcus woesei. FEBS Lett. 407

306:239-242. 408

16. Schut, G. J., S. D. Brehm, S. Datta, and M. W. Adams. 2003. Whole-genome DNA 409

microarray analysis of a hyperthermophile and an archaeon: Pyrococcus furiosus grown 410

on carbohydrates or peptides. J. Bacteriol. 185:3935-3947. 411

17. Shockley, D. R., D. E. Ward, S. R. Chhabra, S. B. Conners, C. I. Montero, and R. M. 412

Kelly. 2003. Heat shock response by the hyperthermophilic archaeon Pyrococcus furiosus. 413

Appl. Environ. Microbiol. 69:2365-2371. 414

18. Bailey, T. L. and Charles Elkan. 1994. Fitting a mixture model by expectation 415

maximization to discover motifs in biopolymers, p. 28-36. In Proceedings of the Second 416

international Conference on Intelligent Systems for Molecular Biology, AAAI Press, 417

Menlo Park, California. 418

19. Bailey, T. L. and Michael Gribskov. 1998. Combining evidence using p-values: 419

Application to sequence homology searches”, Bioinformatics. 14:48-54: 420

20. Trent, J. D., H. K. Kagawa, C. D. Paavola, R. A. McMillan, J. Howard, L. Jahnke, C. 421

Lavin, T. Embaye, and C. E. Henze. 2003. Intracellular localization of a group II 422

chaperonin indicates a membrane-related function. Proc. Natl. Acad. Sci. USA 423

100:15589-15594. 424

21. Trent, J. D., M. Gabrielsen, B. Jensen, J. Neuhard, and J. Olsen. 1994. Acquired 425

thermotolerance and heat shock proteins in thermophiles from the three phylogenetic 426

domains. J. Bacteriol. 176:6148-6152. 427

22. Vierke, G. A. Engelmann, C. Hebbeln, and M. Thomm. 2003. A novel archaeal 428

transcriptional regulator of heat shock response. J. Biol. Chem. 278:18-26. 429


http://jb.asm.org/

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http://jb.asm.org/

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23. Zheng, D., C. Constantinidou, J. L. Hobman, and S. D. Minchin. 2004. Identification 430

of the CRP regulon using in vitro and in vivo transcriptional profiling. Nucl. Acids Res. 431

32:5874-5893. 432


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Table 1. Microarray analysis of whole genome transcription. Whole genome DNA was 433

used as template in cell-free transcription assays. The two reactions containing none or 2.7 434

µM Phr were analysed by microarray experiments. The table shows genes which appeared to 435

be regulated by Phr in multiple of the five independent microarray experiments. 436

437

438


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Figure Legends 439

440

FIG.1. Microarray analysis of whole genome in vitro transcription. The fluorescence 441

intensities of the basal and Phr-regulated transcription are shown. The regulated genes are 442

indicated in red. Whole genomic DNA was used as template in cell-free transcription assays. 443

444

FIG.2. Phr regulates specifically heat shock genes on chromosomal DNA in vitro. Primer 445

extension was conducted to analyze transcription from chromosomal DNA. Lane 1, whole 446

genome transcription without Phr and in presence of 2.7 µM Phr, Lane 2. Analysis of 447

transcripts from phr (A), hsp20 (B), aaa+atpase (C) and gdh (D). 448

449

FIG.3. Confirmation of additional Phr targets by in vitro transcription. A) In vitro 450

transcription results showing repression of six novel genes regulated by Phr. B) Control 451

experiment showing repression of aaa+atpase, phr and hsp20, and no effect on the expression 452

of gdh, which is not regulated by Phr. The reactions contained 0, 50, 100, 150, 200 nM Phr 453

and 10 nM PCR amplified template DNA. Transcriptional activities were quantified using a 454

PhosphorImager. Transcription activity in the absence of Phr was set to 100%. 455

456

FIG.4. Alignment showing the putative binding site of Phr. A) Alignment of all promoter 457

regions of the genes repressed by Phr. The bases that all promoters have in common are 458

shown in red. A difference in one position is labelled in green, two differences in blue, three 459

differences in purple and four differences in yellow. The promoter region of the gene PF1616 460

contains two tandem Phr binding sites (see supplementary Fig. 3) B) Promoter sequences of 461

the genes regulated by Phr. The Phr-binding site is shown in red, the TATA elements are 462

boxed. C) Consensus sequence of an alignment of all promoters using MEME. Adenine is 463

depicted in red, thymine in green, guanine in yellow and cytosine in blue. 464

465

FIG.5. Mutational analyses on Phr and oligonuleotides. Summarized results of mutations 466

on the promoter PF0624. Top panel, EMSA showing binding of Phr to a synthetic 467

oligonucleotide harbouring the consensus Phr binding site (lanes 1,2) and mutations of this 468

sequence as indicated below (lanes 3-10). The presence of Phr in binding reactions is 469

indicated on top of the lanes. Lower panel, sequence of the synthetic oligonucleotide used as 470

template for binding assays and of mutated templates. Bold nucleotides indicate the Phr 471

binding site, and several introduced single point mutations are indicated in red. 472


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Table 1 ORFs which show down-regulation with Phr in a whole genome in vitro transcription.

ORF Interpro name

a Phr observations

b Heat shock

c

PF0624 Conserved hypothetical 4 N PF1042 Ferritin/ribonucleotide reductase-like 2 N PF1291 Phosphoesterase MJ0912 1 N PF1292 conserved hypothetical 2 N

PF1488 Conserved hypothetical 5 N

PF1616 Myo-inositol-1-phosphate synthase (15) 5 Y PF1790 Archaeal heat shock regulator (17, 22) 2 Y PF1882 AAA ATPase (17, 22) 3 Y PF1883 Heat shock protein Hsp20 (17, 22) 5 Y

a Name base on known function or Interpro analysis (http://www.ebi.ac.uk/interpro) b

Number of times the ORF was observed regulated in one of the five independent microarray

experiments. c Regulated by heat shock observed in previous analysis (17, 22)


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1 genome wide identification of targets for the archa...

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